WO1996028967A1 - Transgenic non-primatal mammals wherein serotypes of higher primates have been expressed by foreign gene transfer and method of creating the same - Google Patents

Transgenic non-primatal mammals wherein serotypes of higher primates have been expressed by foreign gene transfer and method of creating the same Download PDF

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Publication number
WO1996028967A1
WO1996028967A1 PCT/JP1996/000703 JP9600703W WO9628967A1 WO 1996028967 A1 WO1996028967 A1 WO 1996028967A1 JP 9600703 W JP9600703 W JP 9600703W WO 9628967 A1 WO9628967 A1 WO 9628967A1
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Prior art keywords
primate
gene
mammal
type
antigen
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PCT/JP1996/000703
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French (fr)
Japanese (ja)
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Chihiro Koike
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Chihiro Koike
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Publication date
Priority claimed from PCT/JP1995/000488 external-priority patent/WO1996028966A1/en
Application filed by Chihiro Koike filed Critical Chihiro Koike
Priority to AU49562/96A priority Critical patent/AU4956296A/en
Priority to PCT/JP1996/000703 priority patent/WO1996028967A1/en
Priority to JP52828796A priority patent/JP3809189B2/en
Publication of WO1996028967A1 publication Critical patent/WO1996028967A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A01K2267/025—Animal producing cells or organs for transplantation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • the present invention relates to a transgenic non-S long-sized mammal having cells, tissues or organs suitable for transplantation into a higher primate by introducing an exogenous gene and a method for producing the animal.
  • the present invention relates to a transgenic animal in which a mammalian glycan antigen is converted into a form unique to a higher primate, and its state is genetically stabilized, and a method for producing the same.
  • kumio includes organs such as bold and liver, as well as skin, blood vessels, and cells.
  • artificial materials such as artificial vessels and artificial blood vessels are used. This includes self tissues, and even tissues and cells of the same or different animal species.
  • Livestock on the other hand, are non-primate mammals, with well-established breeding methods, readily available, and with few ethical issues, making them a promising alternative donor, the so-called donor.
  • tissues isolated from such mammals are known to cause severe rejection in humans, particularly severe rejection that occurs within 24 hours (hereinafter referred to as “super! This is also called a reaction.
  • Such hyperacute rejection occurs in the endothelial glaze within the donor's tissue when the replenished donor tissue vessels are anastomosed with the recipient vessels to flush the blood.
  • This hyperacute rejection reaction in combination with a species in which a human has a natural antibody, is one of the two activation pathways of the human trapping system, one of the classical pathways triggered by the antigen-antibody reaction. It is considered to be the main reaction.
  • This antigen-antibody reaction is thought to be due to the following causes.
  • vascular cells and hemocyte cells containing vascular endothelial cell membranes of non-S-long mammals such as pigs (hereinafter, also simply referred to as vascular cells and hemocyte cells) is high. Because it is different from that of higher primates such as G.
  • non-primate mammals such as septa have their own carbohydrate antigens (hereinafter referred to as G-type antigens or G-type S) that higher primates do not have on their vascular / hemocyte cell membrane surfaces. Because it is.
  • G-type antigens hereinafter referred to as G-type S
  • the anti-G natural antibody in the human blood and the G-type antigen on the vascular endothelial cell membrane in human blood are Antigen-antibody reaction is triggered, and the resulting antigen-antibody complex activates the complement system, causing porcine vascular endothelial cells to rupture, leading to the destruction of the replenished donor tissue, resulting in hyperacute rejection It is.
  • G-type antigen in the donor tissue it is possible to suppress the expression of G-type antigen in the donor tissue by introducing antisense DNA to a gene involved in the expression of G-type antigen in the donor or by completely suppressing the expression of this gene.
  • the GS1 antigen precursor is subjected to SS in the donor male. Since this precursor is also a so-called Bombay-type antigen, it may still cause hyperacute rejection by natural antibodies present in humans.
  • ABH-type substances are expressed in higher primates according to the present invention. Only in rodents to which chimpanzees, orangutans, baboons, Japanese, etc. belong, and in other primates and non-primate mammals, expressed in gastrointestinal muco-knee receptor cells etc. But not expressed in vascular and blood cells.
  • non-primate mammals have a G-type substance in vascular and blood cells that is a substance in which galactose binds to a precursor of ABH-type substance (N-acetyllactosamine) in a 113 structure. is there.
  • the H-type substance also referred to as H-type antigen
  • the H-type antigen is different from the HT precursor (N-acetyllactosamine) in terms of GDP-fucose:) SD-galactoside.
  • G-type substances are the same precursors as the aforementioned H-type substances, N- For cetyl lactosamine, UDP galactose: ⁇ SD-galactosyl-1.4-N-acetyl-darcosaminide 3-galactosyltransferase (hereinafter referred to simply as G-choice or (1.3) galactosyltransferase). It is generated by acting.
  • the difference in serotype between vasculature and blood cells in humans and pigs is due to the difference in enzymes acting on precursors, in other words, the enzyme GT exists in pigs and the like.
  • the enzyme GT does not exist in humans and the like and the enzyme purple FT exists.
  • the presence or absence of ABH-type substances and enzymes in such various tissues is considered to be based on evolution.
  • the present invention provides a non-primate mammal, such as a pig, which is widely bred as a domestic animal and is easily obtainable, by a new method which is not used in the past.
  • the aim is to mitigate rejection and apply non-primate mammals to transplantation into higher primates.
  • the present inventor has proposed that in non-primate mammals such as bushus, gene transfer engineering and developmental engineering, for example, the production of higher-grade glucotransferases such as F-chote.
  • non-primate mammals such as bushus, gene transfer engineering and developmental engineering
  • F-chote higher-grade glucotransferases
  • GT vascular and blood cell
  • the conventional, natural antibodies Attention has been focused on the suppression of the capturer activation pathway after the binding of a protein to an antigen.
  • the present invention can be said to be a more essential solution to the conventional method in that the binding itself between the natural anti-G antibody and the G-type antigen in higher primates such as humans is suppressed.
  • a first aspect of the present invention relates to a non-human mammal having a DNA sequence containing a DNA sequence encoding FT of a higher primate, wherein the DNA construct has been transformed to express an H-type antigen.
  • Transgenic animal In the animal into which this DNA construct has been introduced, FT is expressed, and this FT acts on N-acetyl lactosamine to generate an H-type antigen. Express. For this reason, even if a non-S long mammal has a GT gene, when FT consumes N-acetyl lactosamine, which is a substrate of GT, the non-S long mammal's GT and N-acetyl lactosamine may be intermingled. bond or is inhibited, these foreign DNA beta expression is suppressed G group antigens, be held in a heterozygous state, but may be held at Jo homo-, held in homozygous Jo virtual It is a preferred idea to do so.
  • higher primates refer to narrow monkeys belonging to humans, chimpanzees, orangutans, baboons, Japanese birds, etc. among primates.
  • the non-higher primates are the broad-nosed primates and the protozoa among the primates.
  • Non-primate mammals or non-primate mammals are mammals that are not primates.
  • the second invention comprises a first DNA construct comprising a DNA sequence encoding higher order FT and a second DNA comprising a DNA sequence which is antisense to the GT gene of a non-primate mammal.
  • a non-primate mammal trait characterized in that it has been transformed to express a higher length H-type antigen while reducing expression of a non-primate mammal's G-type antigen. Convertible animal.
  • exogenous DNAs may be maintained in a heterologous state or a homologous state, but it is preferred that they be maintained in a homozygous state.
  • the present invention relates to a method wherein a DNA construct containing a DNA sequence encoding FT is inserted by homologous recombination into a part of the GT gene of a non-primate mammal, thereby expressing a non-primate mammalian G-type antigen.
  • a non-S long mammal transformed animal characterized by being transformed to express a primate H-type antigen.
  • a mutation is introduced into the GT gene of a non-S long mammal by this DNA construct, so that GT is not expressed and G-type antigen is not expressed.
  • FT is expressed, and H-type antigen is expressed by the FT.
  • DNA constructs that cause homologous recombination in part of the GTif gene are:
  • exogenous DNAs may be retained in a hetero state or in a homo state, but are preferably retained in a homo state ffi.
  • a DNA construct containing a DNA sequence encoding FT is inserted into a part of the GT gene of a non-primate mammal by homologous recombination to express a G-type antigen of a non-S long mammal.
  • This is a transplant material obtained from a non-S long mammal transformed animal that has been transformed to express a higher primate H-type antigen without any modification.
  • the cell itself obtained from the transgenic animal, a tissue containing the cell as a component, 3 ⁇ 4, or other materials can be widely used as a material for transplantation into a higher primate.
  • a preferable aspect of the transgenic animal in these inventions is that the non-primate mammal is bush. If the transformed animal is a pig, this can provide material suitable for transplantation into higher primate mammals.
  • a fifth invention is to express a non-primate mammalian G-type antigen by inserting a DNA construct containing a DNA sequence encoding FT into a part of the GT gene of the non-primate mammal by homologous recombination. This is a method for producing a non-primate mammal transformed animal that expresses the H-type antigen of a higher primate.
  • the sixth invention is directed to a method for avoiding rejection reaction between a natural antibody of a higher primate and an antigen of a non-primate mammal in a space for transplanting a tissue of a non-S long mammal into a higher primate.
  • a DNA construct containing a DNA sequence encoding FT is inserted by homologous recombination into a part of the GT gene of a non-primate mammal.
  • H-type of a higher primate without expressing the G-type antigen of the non-primate mammal Creating a non-S long mammal transformed to express the antigen. Transplanting the non-primate mammal progeny obtained in this step into a higher primate.
  • non-primate mammal tissues express the HSS antigen but not the GSS [antigen]. Therefore, when this kumio is transplanted to the higher primate, it is not rejected by the anti-G natural antibody of the higher primate.
  • FIG. 2 is a process diagram of the construction operation of the plasmid pMAM / FT having the FT gene of Example I.
  • FIG. 3 is a schematic view of the fragment for exclusive use of the FT gene of Example I.
  • FIG. 4 is a process (2) of constructing and operating pREP8 / AS / GT of the second embodiment. You.
  • FIG. 5 is a schematic diagram of an AS / GT3 ⁇ 4 gene introduction fragment of Example 2.
  • FIG. 6 is a process diagram of a construction operation of PREP 9 / GT3-4 of the third embodiment.
  • FIG. 7 shows pREP9 / GT3-room SspI nom DraI of Example 3.
  • This figure illustrates the process of assembling I and the process of manufacturing the gene fragment GT 3-/ FT / p 01 yA.
  • FIG. 8 is a process diagram of a construction operation of the pGT / FT of the embodiment S.
  • FIG. 9 is a graph showing the results of 5 , Cr release atsee.
  • FIG. 10 is a diagram showing a method for obtaining fragment A from exons 2 to 3 of G-choedogene.
  • Figure 11 shows a method for obtaining fragment B from exons 4 to 6 of the GT3 ⁇ 4 gene.
  • FIG. 12 is a diagram showing a method for obtaining a plasmid D containing cDNA obtained from exons 1 and 2 of the FT gene.
  • FIG. 1S is a diagram illustrating a method for obtaining a plasmid E from a fragment B and a plasmid D.
  • FIG. 1S is a diagram illustrating a method for obtaining a plasmid E from a fragment B and a plasmid D.
  • Figure 14 shows how to construct from fragment A and plasmid E. 96/28967 N
  • FIG. 1 A first figure.
  • FIG. 15 is a diagram showing a mutant gene when a GT gene and a construct undergo orthotopic recombination.
  • FIG. 16A is a diagram in which the sequence in FT is detected from FT by Southern blotting
  • 16B is a diagram in which the homologous portion of the FT gene on the upstream side is detected by PCR.
  • Fig. 16C shows that 16C confirmed by PCR that homologous recombination occurred in the downstream part of the construct F-finger.
  • 16D shows FT transmission by Northern plotting.
  • Fig. 16 shows that mRNA of the offspring was detected.
  • Fig. 16E shows that D9-29 cells do not express H antigen by D-cytometry using lectin UE A1.
  • Fig. 16F is a diagram showing that the detection of the H antigen of L922 cells into which the FTS gene was introduced by the lectin UE A1 was detected by flow cytometry.
  • 17A is a diagram in which the FT gene was detected by PCR
  • 17B is a PC indicating that homologous recombination was performed in the homologous portion of the construct upstream of FT.
  • R is a diagram
  • 17C is a PCR diagram showing that homologous recombination was performed in the homologous portion downstream of the FT of the construct
  • 17D is an mRNA of the FTS gene.
  • 17E is a flow cytometry diagram showing that L929 cells do not express H antigen using lectin UEA1
  • FIG. FIG. 3 is a diagram showing, by flow cytometry, the detection of H antigen of L929 cells introduced with FT gene into the lectin UEA1.
  • non-primate mammals such as pigs
  • it is an S gene or its expression substance that only higher primates such as humans have and that non-S primate mammals such as septa do not.
  • To express the protein it is necessary to create transgenic animals.
  • blood group matching is one of the basic principles in human-to-human transplantation, but the basic substance of the so-called blood group, or ABO classification, is an H-type substance.
  • the previous substance N Is formed by the action of FT on acetylsilactamine.
  • non-chordate mammals such as pigeons have GT instead of FT, thereby forming a different substance (G-type substance) from human.
  • an exogenous gene fragment containing an FT gene encoding glycosyltransferase FT, which produces an H-type substance from its precursor is introduced into a fertilized egg such as a pig, and this gene fragment is transferred to the chromosome.
  • a fertilized egg such as a pig
  • this gene fragment is transferred to the chromosome.
  • Non-II long mammals expressing H-type K have similar acute serotypes in their blood and blood serotypes, and hyperacute rejection when transplanting the tissues of these animals into higher primates. The response can be mitigated, making these animal tissues suitable for transplantation into higher primates.
  • the enzyme FT consumes the precursor ⁇ , it is expected that the binding of the endogenous enzyme GT to the precursor will be inhibited and the production of type G substances will be suppressed.
  • the FT3 ⁇ 4 gene can be stably transmitted to progeny after it has been incorporated into the chromosome.
  • the present invention inactivates the expression of glycosyltransferases endogenous in non-primate mammals such as pigs, for example, the gene transfer of the plant transferase GT, which produces a G-type substance from its precursor.
  • a foreign gene fragment containing the antisense DNA of the GT G gene is introduced into the fertilized egg.
  • this gene fragment is integrated on the human chromosome, if this antisense DNA is transcribed in each cell, the transcript will be paired with the endogenous GT transcript and the GT It is expected that translation of offspring will be suppressed.
  • the enzyme F is actively expressed, the expression of the enzyme GT is suppressed, and H-type substances are produced, while production of G-type substances is reduced. Evening can be obtained.
  • foreign gene fragments such as antisense DNA can be stably transmitted to progeny after being integrated into the chromosome.
  • the foreign gene Once the foreign gene has been integrated into the chromosome, it can be homogenized by mating with the foreign gene, and the foreign gene that expresses the transferase of higher primates can be obtained.
  • higher primate sugars can be used.
  • a non-primate mammalian system that expresses a transferase and exhibits a higher serotype serotype can be obtained.
  • humans can be particularly targeted as higher primates.
  • Higher S-long bran transferase refers to a glycosyltransferase that is found in higher S-longs and contributes to the production of antigens not found in non-S-long mammary vascular and blood cells.
  • a specific example is FT.
  • the human FT is [EC 2, 4.1.69].
  • a DNA construct containing a DNA sequence encoding a higher length bran transferase can have a promoter, a terminator and the like in addition to the DNA sequence of the gene for the glycosyltransferase.
  • the terminator associated with the Boli A addition signal of the initial gene of SV40 is one of the sequences suitable for addition.
  • the expression level of the transgene is considerably influenced by the B-A signal taken from which gene, the B-A addition from the initial SV40 gene is not I'm good. This signal is placed before the terminator.
  • To form a foreign gene that can express FT it is necessary to know at least the DNA sequence of FT. Human FT has already been reported (Proc. Tl. Acad. Sci. USA. 87 (1990), pp ⁇ 67 4-6678).
  • non-vertebrate mammal in particular, an animal to be subjected to xenotransplantation to the higher primate can be targeted.
  • stags, puppies, sheep, goats, etc. raised as domestic animals are suitable.
  • These livestock are easy to breed and can be supplied stably, so they are suitable for replenishment of transplanting males.
  • Glycosyltransferases in non-primate mammals are ⁇ transferases involved in the production of antigens found in the vascular and blood cells of non-primate mammals and not found in higher primates.
  • a specific example is the pig-to-mouse GT.
  • the mouse GT is specified in [EC 2.4.1.15.1].
  • DNA constructs that suppress the expression of enzymes include DNA constructs containing a DNA sequence that is antisense to the DNA sequence that encodes the glycosyltransferase.
  • a DNA construct containing a DNA sequence that disrupts DNA can be used. To form this DNA construct, it is necessary to know at least the non-primate glycosyltransferase gene sequence.
  • the mouse GT gene has already been reported (Proc. Natl. Acad. Sci. USA. (1989), pp. 8227-8231). Also, the GTA gene of buta was reported to K (Xe notransplantation).
  • a DNA fragment in which a part or all of the cDNA of GT gene is incorporated in the antisense direction is formed as a DNA construct.
  • homologous recombination for example, it is preferable to form a region having as many regions of homology with the GT gene as possible.
  • glycosyltransferase in non-primate mammals can be suppressed by a conventionally known method other than introduction of a foreign gene.
  • the DNA construct can be constructed not only for non-homologous recombination on the chromosome but also for homologous recombination.
  • the pig FT gene is recombined with the pig GTit gene as a target, the suppression of GT expression and the expression of FT can be simultaneously performed.
  • a means for expressing the glycosyltransferase at a high rate when the gene fragment is recombined into a chromosome can be used.
  • a foreign gene fragment is prepared using pMAM as a vector plasmid, high expression can be induced by administration of steroid.
  • non-primate mammal There are many methods for introducing an exogenous gene into a non-primate mammal, such as a method using a retrovirus and a method using microinjection, and any method can be adopted. From the viewpoint of obtaining a progeny transformation when the t it is desired to germline transformation, non-primate mammalian embryo ⁇ or ES cells, or, is preferably performed with respect to a fertilized egg.
  • ⁇ -transferase Due to the expression of ⁇ -transferase in higher primates, or at the same time, the expression of ⁇ ⁇ -transferase in non-prolonged mammals is suppressed, the animal has a blood serotype similar to that of higher primates. Therefore, it is possible to mitigate the acute rejection reaction when these animal tissues are transplanted into higher S primates, and these animal tissues are suitable for transplantation into higher primates. It will be. In particular, when expressed in vascular endothelial cells, hyperacute rejection is greatly reduced. In addition, the expression of sugar priming enzymes in higher primates in red blood cells makes the serotype of blood similar to higher primates, and the blood of these animals is suitable as a substitute for higher S primates.
  • the H-type substance is expressed and the G-type substance is simultaneously reduced in the vascular and blood cell cells of non-long-term mammals.
  • Tissues will be animals that are similar to blood serotypes or higher primates.
  • the G-type substance is further reduced, and the animal becomes an animal whose tissue and blood serotypes are very similar to those of higher vertebrates.
  • FT when FT is expressed on the surface of vascular endothelial cells, hyperacute rejection in xenogeneic organs or tissue transplantation is greatly reduced. Also, if expressed in erythrocytes, it will be more suitable for blood substitutes.
  • one or more of the GT G gene loci on the chromosome do not exist, and FT
  • at least one GT exists on the chromosome, so that an individual with FT or two GTs can be obtained instead.
  • Such an individual provides a transplant material suitable for transplantation of a human or the like as a donor, regardless of the recipient's ABO blood type.
  • such a transformed non-primate mammal has a tissue that can be transplanted into a higher primate such as a human, and thus becomes a source of a substitute for a tissue such as a human because it has blood.
  • these animals are stably bred, they will be able to provide necessary pheasants and the like when needed, and will be used as storage for transplanted tissues.
  • a method to provide animals and materials that can mitigate hyperacute rejection in the case of xenograft ra transplantation by expressing the FTS gene specific to higher S-length mammals in non-S-length mammals is an unprecedented new method. And provide a more essential solution for the reconciliation of hyperacute rejection in xenotransplantation.
  • a sugar chain antigen of a higher primate is produced in its tissue and blood, or simultaneously, the production of a sugar chain antigen of a non-primate mammal is suppressed.
  • hyperacute rejection can be mitigated when non-s long mammal tissues are transplanted into higher primates, which are allogeneic transplants, and can be replaced with conventional artificial blood vessels, artificial organs, or allogeneic transplants. It is possible to provide a material that can be used in place of a magnetic device, a magnet assembly, and the like.
  • transforming a non-mammal mammal according to the present invention does not cause any damage or abuse to these animals. It does not affect the survival, reproduction, etc. of the fish. Further, the present invention does not exert any adverse effects on humans or any other animals.
  • examples of the present invention will be described.
  • the exogenous s gene in the present invention the two types of ⁇ -transferases already described, namely the G Tit gene and the cDNA of the F T gene, can be exemplified. However, the following embodiment does not limit the IS area of the claim.
  • MCS multicloning site
  • pMAM a product of CL0NETECH, Lee.F. (1981) Nature 294: 2278
  • restriction enzymes Nhel and Xhol restriction enzymes Nhel and Xhol
  • LMP gel LMP gel
  • the purified FT gene cDNA was inserted into this vector using T4 DNA ligase.
  • the plasmid DNA was used to transform JM109 bacterial cells in a combinatorial concept, and the resulting transformants were selected on an Ambicillin plate.
  • Each individual colony was allowed to grow with an LB medium (composition: Bacto-tryprone 10 g (manufactured by DIFC0), Bacto-yeast extract 5 g (manufactured by DIFC0), and NaCl 10 g to make 1 L with water, the same applies hereinafter).
  • a mini-plasmid was prepared. The extracted plasmid DNA was digested with various restriction enzymes and subjected to electrophoresis to spot the size and position of the inserted fragment. As a result, the plasmid integrated in the correct direction was called pMAMZ FT (see 02).
  • E. coli JM109 / p was used to obtain a linear fragment for gene transfer.
  • Plasmid preparation was performed after mass-culturing the MAM / FT strain in LB medium. Further, the obtained plasmid was oval by CsC1 density gradient centrifugation. After digestion of 10 g of the coffin-produced brasmid with the restriction enzymes PVu] and BamHI, this fragment was purified and separated to obtain a fragment for FTS gene transfer. A schematic diagram of the construction of this fragment is shown in FIG.
  • RNA was extracted, and cDNA corresponding to total RNA was synthesized using reverse transcriptase. This cDNA was transformed into type III, and PCR was performed using the primers described above.
  • the cDNA fragment obtained by this PCR was subjected to 1% agarose gel electrophoresis, a band having a length of about llOObp was obtained.
  • the base sequence was read by automatic base sequence reading (1), and it had the base sequence of the GT gene as previously reported. . Therefore, the cDNA was purified by electrophoresis on an LMP gel, treated with restriction enzymes Nhel and Xhol, and then separated and distributed using a Wizard system.
  • Escherichia coli JM109 / pREP8ZAS / GT strain was mass-cultured in LB medium to prepare a linear fragment for gene introduction, and then plasmid was prepared. Further, the obtained plasmid was purified by CsC1 density gradient centrifugation. After digesting 10 g of the purified plasmid with the restriction enzymes XbaI and PstI, this fragment was purified and separated by LMP gel electrophoresis and the Wizard system, and then used as a fragment for AS / GT gene transfer. did. The construction scheme of this fragment is shown in FIG.
  • Plasmid containing plasmid for homologous recombination (pREP 9ZGTZFT) and preparation of gene fragment for transfection (PG-cho / FT)
  • a primer with a restriction enzyme Kpn I cleavage site (referred to as ⁇ / ⁇ ) as a sense primer in exon 3 and an antisense primer A primer (pZB) containing a restriction enzyme Ss ⁇ I cleavage site at the start codon site in exon 4 and a restriction enzyme HindIII cleavage site downstream thereof was synthesized.
  • the PCR method was performed using these primers p / A and p / B, and the obtained fragment was cut with restriction enzymes KpnI and HindIII, purified and separated, and this was separated into GT3- Called 4.
  • the expression vector (pREP 9 Unvitrogen) is cut with the restriction enzyme BamHI, the end is blunted with T4 DNA polymerase, self-ligated with T4 DNA ligase, and the restriction enzyme BamHI is used.
  • the expression vector from which the cleavage site was deleted was designated as pREP9 / amBamHI.
  • the expression vector was called pREP 9ZABamH I Nom Dr a III. This vector was digested with restriction enzymes KpnI and HindIII, and the plasmid into which the above GT3-4 was inserted was designated as PREP9ZGT3-4.
  • PCR method was performed using these primers, the resulting fragment was digested with restriction enzymes NheI and XhoI, and pREP9 / GT3-4 was digested with restriction enzymes NheI and XhoI. Insert the cut plasmid. This was called pREP 9ZGT 3-4ZFT.
  • pREP 9ZGT 3-4ZFT When this plasmid PREP / GT3-4ZFT was cleaved with restriction enzymes SspI and DraIII, blunt ends were generated.
  • both ends were linked by sdf-ligation, and the FT cDNA expression initiation site was located at the GT expression initiation site.
  • This brassmid was designated as pREP9 / GT3-4 /? C / m3spI / ⁇ railI.
  • these restriction enzymes KpnHindl! SspI, Nhe], DralII, and XhoI were all fragments from exon 3 to exon 4 and the expression site of FTcDNA. It has already been found not to cut fragments.
  • An antisense primer (p / E) was synthesized containing the 3 'side of p01yA of PRE P9 and containing the restriction enzyme BamHI cleavage site, and a sense primer (P / A) And pREP 9 / GT3-4 / FT / AS sp I / AD ra
  • the fragment was purified by cutting with HI and separated, and the obtained fragment was designated as GT3-4 / FTZpolyA (see FIG. 7).
  • the expression vector pREP9 was digested with restriction enzymes KpnI and BamHI, and the above fragment GT3-4 / FT / p01yA was introduced. This is pREP 9ZGT3
  • the exon 4 to exon 6 part of the GT was digested with the restriction enzyme BamHI site for both the sense primer (pZF) and antisense primer (p / G), and extracted by PCR.
  • the fragment obtained by digestion with the restriction enzyme BamHI was inserted into a plasmid pREP9 / GT3—4noFTZpo1yA that had been opened with the restriction enzyme BamHI (BamHI was GT It has already been ascertained that 3-4, FT, po 1 y A and T 4-7 do not cut).
  • the resulting plasmid was designated as PREP 9ZGT3-4 / FTZpolyolAZGT4-6, or simply as pREP9 / GTZFT.
  • the plasmid was subjected to PCR using primers p / A and p / G.
  • the fragment obtained after purification was used as a fragment for introduction into a fertilized egg, and was referred to as GT3-4 / FT / p01yA / GT416, or simply pGTZFT.
  • the transgene fragment for introduction thus prepared has high homology with the gene of the GT chromosome and is expected to undergo homologous recombination.
  • the individual (F o) expresses both GT and FT because F-cho is expressed instead of GT.
  • a valence having only FT can be obtained according to Mendel's law. This is the transgenic animal that has no antigen specific to non-primate mammals but has an antigen specific to higher primates. Since the homology between mouse GT and Busu GT is high, the homologous recombination gene based on mouse GT can be applied to pigs.
  • the solution containing the two fragments for transfection is prepared by adding these transfection fragments to a buffer solution (0.25 mM EDTA, 5m Tris (pH 7.4)), and adding 1.0 gZml each.
  • a buffer solution (0.25 mM EDTA, 5m Tris (pH 7.4)
  • the 'fertilized egg was transplanted to the oviduct of a female adult pigs that estrus. Transplantation was performed by intratubal transplantation after laparotomy under general anesthesia. After tubal transplantation, the animals were bred to term until delivery, and pups were obtained.
  • the fertilized eggs into which the gene has been injected are preferably transplanted immediately.
  • cDNA was synthesized from total RNA by reverse transcriptase, and PCR was performed using the primer used for FT gene extraction described above. As a result, one of 27 animals was determined to be 13 ⁇ 4. That is, it was confirmed that the introduced FT gene was translated into mRNA and produced an FT enzyme protein. A similar search was performed on 15 control pigs into which no gene had been introduced, but all were negative.
  • Example 6 It was determined to be positive in Example 6, that is, it is considered to be producing FT protein Blood was collected from the pupa pupa, and after the heart, blood cells were separated and swine blood cells were stained by the FITC method using a fluorescent antibody. On the other hand, blood was also collected from control pigs (producers without transgenes) and stained similarly.
  • an anti-H-type antibody was used as a primary antibody, and mouse 7 globulin (F (ab) 2 ) labeled with a fluorescent dye was used as a secondary antibody.
  • F (ab) 2 mouse 7 globulin labeled with a fluorescent dye
  • the so-called blood type is converted, and as a result, the same H-type substance as the human type 0 is produced. It was shown that the antibody did not form an antigen-antibody reaction with the natural antibody, or that the formation of the antigen-antibody reaction was infrequent.
  • fragment A fragment A (fragnientA) is described below with reference to Fig. 10.
  • fragment A was cleaved with various restriction enzymes including the restriction enzyme Sphl, and it was found that there was 1 Sf of the restriction enzyme Sphl cleavage site at about 4.81 cbp upstream of exon 3. Then, the plasmid A was cleaved with restriction enzymes Spill and Sail to obtain a fragment of about Slcbp. After purification and separation, the fragment was blunt-ended using T4 DNA polymerase (Takara Shuzo). This was called fragment A (fr3 ⁇ 4ineiitA).
  • fragment B (irftgm e nt B)
  • Brasmid J> GEM-T Promega
  • Brasmid B piumid B
  • fragment B (plasmid B) was found to be restricted to the cloning site on the 5 'side of the plasmid pGEM-T (Promega). The fragment was cut with NcoI to obtain a fragment of about 3. bp. After this was isolated from the manufacturer, the above fragment was blunt-ended using T4 DNA polymerase (Takara Shuzo). To this fragment was added Sac linker (New Eflland Bioiab nc.), which was filled with nucleotides. This was called fragment B (fragmem B).
  • this fragment (fra ment ed. Incorporated into an expression vector pCR3 (Invitrogen, Inc.), was designated At the bra corner de C (plasmid C) t
  • the above expression vector The only restriction enzyme ⁇ 11 is present at nucleotide position 1113 of pCR3, that is, immediately downstream of the poiy A site of pCR3.
  • This restriction enzyme is a fragment C containing FT (frtgment C ) Is cut off. Therefore, plasmid C is restricted with restriction enzyme C. Cut, this before
  • Brasmid E (piasmidE) is described below with reference to FIG.
  • the plasmid E was cut with the restriction enzyme Sail, blunt-treated, and ligated with the fragment A (ir meiitA) using the DNA ligese (Takara Shuzo).
  • the above plasmid F was cut with restriction enzymes Sai I and Ail III. This resulted in a fragment approximately 10 kbp in length. This was manufactured as described above, and the portability was adjusted to 5.0 ⁇ 8 / ⁇ 1; as a ⁇ gene fragment for introducing g gene, it was referred to as ⁇ / ⁇ .
  • this construct has a mutant sequence inserted in a form that deletes the sequence portion containing the translation initiation code of the .GT gene.
  • this inserted sequence Contains all the translation parts (including the translation start code and translation stop code) that encode the FT Liao gene.
  • the indented sequence is the .FT translation part. 3 'is to include a terminator.
  • the god-introduced array is a translator of GT's ⁇ ⁇ ⁇ It is linked to the untranslated part on the 5 'side of the ⁇ code.
  • Fifth. The length of the part of the inserted sequence is reduced. In other words, the length of the sequence of the construct is almost the same as the length of ⁇ that causes homologous recombination in the existing gene of GT.
  • homologous recombination is relatively likely to occur. If the homologous recombination force occurs, the GT will be expressed due to the lack of the translation start code in the GT igi cloud, and the FT will follow The child is translated and expressed.
  • FCS blood culture medium
  • RPMI RPMI until about 1.0x10 s cells are observed, and this day is the first day.
  • change to a serum-free medium and after about 3 hours The following solutions were added to the culture dishes. That is, a mixed solution was prepared by adding 2 ⁇ l of the gene fragment GT / FT and 7 ⁇ l of the pMAM w (l. ( ⁇ G ⁇ i) to the 7 ⁇ l DMEM solution to the 7 ⁇ l DMEM solution. In the DMEM solution,
  • the medium was replaced with S at 1 O ml, and the culture was further cultured for 7 days.
  • the medium was replaced with 10 ml of the PRMI solution with 1095 FBS and 10 ml of G418.
  • the above-mentioned transduced cells were further cultured.
  • the total NA was extracted.
  • RT-PCR was performed using the above primers and F.
  • three out of the three cell groups were subjected to RT-PCR! It was positive (Fig. 16D).
  • the exogenous gene group and the non-transfected cell group were stained with a lectin (UEA-1; EY bolatones) labeled with 3 ⁇ 4IS, and the expression of the H antigen was determined by flow cytometry. Confirmed ( Figures 16E and F).
  • FIG. 16E indicates that the expression level of H antigen was measured using the lectin UEA-I in a mouse LS29 cell group into which the neomycin-resistant ifi gene was introduced.
  • FIG. 1 shows L929 I FT ⁇ UEA-I of I5F. The degree of H antigen expression was measured using the lectin UEA-I in the mouse L929 cell group into which the exogenous pathogen L-GT / FT was introduced. Indicates that
  • TTGTGCCAQC AGTTTTCTGA ATTT6AAAGA GTATTACTCT GGCTACTTCC 118G
  • CB nucleic acid

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Abstract

The hyperacute rejection occurring in the transplantation of tissues of a non-primatal mammal into a higher primate can be mitigated by transferring foreign genes of a higher primate, which express a sugar transferase, into a non-primatal mammal so as to express sugar-chain antigens of the higher primate.

Description

明紬害 外来遣伝子の導入により高等 I 長類の抗原型を発現した非霊長哺乳類の形質転換 動物及びその作出方法  Transformation of non-primate mammals expressing higher I serotype by introduction of foreign gene
〔技術分野〕  〔Technical field〕
この発明は 外来遗伝子導入により、 高等霊長類への移植に適した細胞、 組織あるいは臓器等を有する非 S長晡乳類の形質転換動物及びその動物の作出方 法に関し、 特に、 非霊長哺乳類の糖鎖抗原を高等霊長類に特有な形に転換し、 そ の状態を遣伝的に安定化させた形質転換動物及びその作出方法に閻するものであ る。  The present invention relates to a transgenic non-S long-sized mammal having cells, tissues or organs suitable for transplantation into a higher primate by introducing an exogenous gene and a method for producing the animal. The present invention relates to a transgenic animal in which a mammalian glycan antigen is converted into a form unique to a higher primate, and its state is genetically stabilized, and a method for producing the same.
〔背景技術〕  (Background technology)
今日、 ヒ トの欠損あるいは不完全な組総を、 代替物を移植することにより 補充する治療法か一股的な治療法の一つとなりつつある。 ここで、 例えば組雄と は、 晋胆や肝臓等の臓器から、 皮膚、 血管、 細胞までも含まれ、 かかる組維の代 替物としては、 人工ほ器や人工血管に代表される人工材料、 自己の組維、 さらに は同種あるいは異種の動物の組織や細胞までか含まれるものである。  Today, human replacement or replacement of missing or incomplete human populations by transplantation is becoming one of the most promising treatments. Here, for example, kumio includes organs such as bold and liver, as well as skin, blood vessels, and cells. As an alternative to such tissue, artificial materials such as artificial vessels and artificial blood vessels are used. This includes self tissues, and even tissues and cells of the same or different animal species.
しかし、 人工臓器の多くは、 いまのところ人工弁の他は移植された動物体内で 臓器として充分に機能できるまでには至っておらず、 また、 人工血管においては、 その内部に血栓が生じやすいという欠点があり、 とりわけ、 人工血管の直径か小 さいほど、 その傾向が大きいという問鼸がある。  However, most artificial organs have not yet functioned sufficiently as organs in transplanted animals, except for artificial valves, and artificial blood vessels tend to have blood clots inside them. There is a drawback, especially the smaller the diameter of the vascular graft, the greater the tendency.
また、 自己の組維を代替物として用いるにあたっては、 補充用として利用可能 な器官や量に極めて限りがあるし、 かかる組織を摘出し、 さらに補充するための 身体への負担を考慮すれば、 実際には不可能なことも多い。  Also, when using your own tissue as a substitute, the organs and quantities available for replenishment are extremely limited, and given the burden on the body to remove and replenish such tissue, There are many things that are impossible in practice.
さらに、 ヒ トに近い高等盖長類から摘出した組織により補充するには、 その飼 育上の間題から需要に応じ切れないとともに、 倫理的観点等からの問題があるた め、 困難なことが多い。  Furthermore, it is difficult to replenish with tissues extracted from higher caps close to humans, because it is difficult to keep up with demand due to the problem of breeding, and there are problems from the ethical point of view. There are many.
一方、 家畜は、 非靈長哺乳類であって、 その飼育方法が確立しており、 容 易に入手でき、 倫理的問題も少ないため、 代替物の提供体、 いわゆる ドナーとし て有望である。 しかしながら、 その反面、 かかる哺乳動物から摘出した組繳は、 ヒトにお いては瀲しい拒絶反応を引き起こすことが知られ、 特に 2 4時間以内に発生する 激しい拒絶反応 （以下、 超 !&性拒絶反応ともいう。 ） が問¾となっている。 Livestock, on the other hand, are non-primate mammals, with well-established breeding methods, readily available, and with few ethical issues, making them a promising alternative donor, the so-called donor. However, on the other hand, tissues isolated from such mammals are known to cause severe rejection in humans, particularly severe rejection that occurs within 24 hours (hereinafter referred to as “super! This is also called a reaction.
かかる超急性拒絶反応は、 補充したドナー組織の血管をレシピエントの血管と を吻合して血液を港流する際に、 ドナーの組纔内の血管内皮釉胞で生じる。  Such hyperacute rejection occurs in the endothelial glaze within the donor's tissue when the replenished donor tissue vessels are anastomosed with the recipient vessels to flush the blood.
この超急性拒絶反応は、 ヒ 卜が自然抗体を持つところの種との組み合わせ においては、 2種類あるヒ トの捕体系の活性化経路のうち、 抗原抗体反応によつ て引き起こされる古典経路が主要な反応であると考えられている。 この抗原抗体 反応は、 以下の原因によるものと考えられている。  This hyperacute rejection reaction, in combination with a species in which a human has a natural antibody, is one of the two activation pathways of the human trapping system, one of the classical pathways triggered by the antigen-antibody reaction. It is considered to be the main reaction. This antigen-antibody reaction is thought to be due to the following causes.
まず、 第 1 に、 ブタなどの非 S長類哺乳動物の血管内皮細胞膜を含む脈管 系細胞及び血球系紬胞 （以下、 単に脈管，血球系細胞ともいう。 ） 上の抗原性が ヒ ト等の高等霊長理のそれと異なるからである。  First, antigenicity on vascular cells and hemocyte cells containing vascular endothelial cell membranes of non-S-long mammals such as pigs (hereinafter, also simply referred to as vascular cells and hemocyte cells) is high. Because it is different from that of higher primates such as G.
すなわち、 プタなど非霊長哺乳類は、 高等霊長類の持っていない彼ら特有 の糖鎖抗原 （以下、 この抗原を G型抗原あるいは G型物 Sという。 ） をその脈管 •血球系細胞膜表面に持っているからである。  In other words, non-primate mammals such as septa have their own carbohydrate antigens (hereinafter referred to as G-type antigens or G-type S) that higher primates do not have on their vascular / hemocyte cell membrane surfaces. Because it is.
第 2に、 ヒ トはブタなどの G型抗原に対する自然抗体を生まれながらに持って いるか であ o。  Second, do humans naturally have natural antibodies against G-type antigens such as pigs?
したがって、 例えばプタをドナーとする場合に、 ブタの血管の中にヒ トの 血液が流された際、 ヒ トの血液中の抗 G自然抗体とブ夕の血管内皮細胞膜上の G 型抗原との抗原抗体反応が引き起こされ、 その結果形成された抗原抗体複合体が 補体系を活性化し、 ブタの血管内皮紬胞が破壤され、 補充したドナー組織の破壊 につながり、 超急性拒絶反応か生ずるのである。  Therefore, for example, when using a septum as a donor, when human blood is flowed into a pig blood vessel, the anti-G natural antibody in the human blood and the G-type antigen on the vascular endothelial cell membrane in human blood are Antigen-antibody reaction is triggered, and the resulting antigen-antibody complex activates the complement system, causing porcine vascular endothelial cells to rupture, leading to the destruction of the replenished donor tissue, resulting in hyperacute rejection It is.
かかる超急性拒絶反応を回避するために、 非霊長哺乳類の組截を摘出して ヒ トに補充する際に、 同時にヒ ト等のレシビエン トにおける補体の活性を制限す る物質を供給する技術も開示されているが （特表平 5 - 5 0 3 0 7 4号公報 0 9 1 / 0 5 8 5 5 ) 、 そこに示されている補体制限因子を持 όたブタの細胞と ヒ トの血流との関係は、 基本的にはいわゆる異型輪血と同じ構造を持った関係と なるため、 超急性拒艳反 の発生を必ずしも防止できるものではなく、 又、 ヒ ト が動物の抗原に豚作されるため、 いわゆる血清病を誘発する可能性を併せもっと いう不都合がある。 In order to avoid such super-acute rejection, a technique for supplying a substance that restricts the activity of complement in a recipient such as a human at the same time as excision of a non-primate mammal and replenishing the human with the same. (Japanese Patent Application Laid-Open No. 5-53074 / 09/0855/55), however, discloses that pig cells and humans having complement restriction factors indicated therein are disclosed. The relationship with the blood flow of the animal is basically a relationship having the same structure as the so-called atypical ring blood, so that it is not always possible to prevent the occurrence of hyperacute rejection, and that the Because pigs are made with antigens, the possibility of inducing so-called serum sickness is added. There is an inconvenience.
また、 ドナーにおいて G型抗原の発現に係わる追伝子に対するアンチセン ス DNAを導入したり、 この遗伝子の発現を完全に抑制したりしてドナー組維に おける G型抗原の発現を抑制しょうとする考えもある （WO 94/ 2 1 79 9 ) 。 しかし、 この方法だけでは、 ドナー組雄に、 GS1抗原の前駆物質が SSされるこ とになる。 この前駆物質は、 また、 いわゆるボンベイ型抗原であるため、 依然と してヒ トに存する自然抗体による超急性拒铯反応を生じる場合がある。  In addition, it is possible to suppress the expression of G-type antigen in the donor tissue by introducing antisense DNA to a gene involved in the expression of G-type antigen in the donor or by completely suppressing the expression of this gene. (WO 94/2179 9). However, with this method alone, the GS1 antigen precursor is subjected to SS in the donor male. Since this precursor is also a so-called Bombay-type antigen, it may still cause hyperacute rejection by natural antibodies present in humans.
さらに、 同種移植の場合は免疫抑制剤の利用は有効な場合か多いが、 異種移植 の場合は多剤併用してもなお、 今日、 超急性拒艳反応を免れていない。  Furthermore, in the case of allogeneic transplantation, the use of immunosuppressive drugs is often effective, but in the case of xenograft transplantation, even with the combination of multiple drugs, hyperacute rejection has not been spared today.
ここて、 脈管 ·血球系細胞における ABH型物質の異種間分布をみると、 ABH型物質が発現されるのは、 本発明にいう、 高等霊長類、 すなわち、 真笾亜 目のうち、 ヒ トゃチンパンジー、 オランウータン、 ヒヒ、 日本瘼などが属する狭 袅摟類だけであって、 他の霊長類や非霊長類哺乳動物では消化管粘膝ゃ唉覚受容 体細胞などでは発現されていることがあるが、 脈管 ·血球系細胞には発現されて いない。  Here, looking at the cross-species distribution of ABH-type substances in vascular and hemocyte cells, ABH-type substances are expressed in higher primates according to the present invention. Only in rodents to which chimpanzees, orangutans, baboons, Japanese, etc. belong, and in other primates and non-primate mammals, expressed in gastrointestinal muco-knee receptor cells etc. But not expressed in vascular and blood cells.
一方、 非霊長哺乳類が脈管 ·血球系細胞に有する G型物質は、 ABH型物 質の前駆物質 （N—ァセチルラク トサミ ン） に対し、 ガラク トースがな 1 一 3構 造で結合した物質である。  On the other hand, non-primate mammals have a G-type substance in vascular and blood cells that is a substance in which galactose binds to a precursor of ABH-type substance (N-acetyllactosamine) in a 113 structure. is there.
A BH型物質と G型物質の合成経路について図 1に示す。  Figure 1 shows the synthetic pathways for ABH and G-type substances.
図 1から明らかなように、 本発明にいう H型物質 （H型抗原ともいう。 ） は HT 駆物質 （N—ァセチルラク トサミ ン） に対して、 GDP- いフコース： ）S-D- ガ ラク トシ ド 2- な- レフコシルトランスフェラーゼ （以下、 単に FTあるいは （ 1, 2) フコシル トランスフ Iラーゼという。 ） が作用して生成され、 G型 物質は、 前述の H型物質と同じ前駆物質である N—了セチルラク トサミ ンに対し て UDPガラク トース ： ^S-D- ガラク トシル -1.4- N- ァセチル ダルコサミ ニ ド 3- ガラク トシルトランスフェラーゼ （以下、 単に G丁あるいはな ( 1. 3) ガラク トシル トランスフェラーゼという。 ） が作用して生成されるの である。 このように、 ヒ ト等とブタ等における脈管，血球系钿胞における抗原型 の差異は、 前駆物質に作用する酵素の差異、 換言すれば、 ブタ等に酵素 GTが存 在し、 反対にヒ ト等に酵素 G Tが存在せず酵紫 F Tが存在するという差異に基づ く ものである。 このような各種組織における A B H型物質の存否、 酵素の存否は、 進化に基づく ものであると考えられている。 As is evident from FIG. 1, the H-type substance (also referred to as H-type antigen) according to the present invention is different from the HT precursor (N-acetyllactosamine) in terms of GDP-fucose:) SD-galactoside. It is produced by the action of 2-na-lefucosyltransferase (hereinafter simply referred to as FT or (1,2) fucosyltransferase), and G-type substances are the same precursors as the aforementioned H-type substances, N- For cetyl lactosamine, UDP galactose: ^ SD-galactosyl-1.4-N-acetyl-darcosaminide 3-galactosyltransferase (hereinafter referred to simply as G-choice or (1.3) galactosyltransferase). It is generated by acting. Thus, the difference in serotype between vasculature and blood cells in humans and pigs is due to the difference in enzymes acting on precursors, in other words, the enzyme GT exists in pigs and the like. On the contrary, it is based on the difference that the enzyme GT does not exist in humans and the like and the enzyme purple FT exists. The presence or absence of ABH-type substances and enzymes in such various tissues is considered to be based on evolution.
しかしながら、 かかる高等 長類と非霊長類哺乳動物におけるそれぞれ特 有の链 «抗原の分布と、 超急性拒絶反応の主たる場である血管内皮細胞等におけ るこれら糖鎖抗原の差異を！！極的に利用して、 超急性拒絶反応を雄和することに ついては、 従来全く着目されていなかった。  However, the unique distribution of these antigens in higher order and non-primate mammals and the difference between these carbohydrate antigens in vascular endothelial cells and the like, which are the main sites of hyperacute rejection, have been demonstrated! ! In the past, there has been no focus on the use of extreme acute rejection to harmonize.
〔発明の開示〕  [Disclosure of the Invention]
そこで、 本発明は、 従来の異種間移植における問題を解決するべく、 家畜 として広く飼育され入手の容易なブタ等の非霊長類哺乳動物において、 従来にな い新しい手法により、 移植時の超急性拒絶反応を緩和して、 非霊長類哺乳動物の 組繳を高等簠長類への移植に適用することを目的とするものである。  In order to solve the problems in conventional xenotransplantation, the present invention provides a non-primate mammal, such as a pig, which is widely bred as a domestic animal and is easily obtainable, by a new method which is not used in the past. The aim is to mitigate rejection and apply non-primate mammals to transplantation into higher primates.
上記目的を達成するため、 本発明者は、 ブ夕等の非霊長類哺乳動物におい て、 遣伝子工学的 ·発生工学的に、 例えば F丁等の高等蓥長類の糖転移酵素を発 現させて、 高等^長類の抗原物質を積極的に生成せしめ、 あるいは同時に、 例え ば G T等の非霊長類哺乳動物の糖転移酵素の発現を抑制して、 脈管 ·血球系紬胞 を抗原的に高等霉長類型に転換することにより、 高等霊長類に非霊長類哺乳動物 の組維を移植した際の超急性拒艳反応を锾和できることを見い出し、 以下の発明 を完成したのである。  In order to achieve the above object, the present inventor has proposed that in non-primate mammals such as bushus, gene transfer engineering and developmental engineering, for example, the production of higher-grade glucotransferases such as F-chote. To actively generate higher ^ length antigenic substances, or at the same time, suppress the expression of glycosyltransferases in non-primate mammals such as GT, thereby reducing vascular and blood cell It was found that by converting to a higher primate type antigenically, hyperacute rejection when transplanting a non-primate mammal tissue into a higher primate was alleviated, and the following invention was completed. .
なお、 異棟間移植の超急性拒絶反^を緩和させるために、 ドナーにおいて ヒ ト等の高等霊長類とおなじ H型物質を発現させることは従来考えられていない c すなわち、 従来は、 自然抗体と抗原との結合後における捕体活性化経路の抑制等 に着目されていた。 また、 本発明は、 ヒ 卜等の高等霊長類における抗 G自然抗体 と G型抗原との結合自体を抑制する点で、 従来の手法に対し、 より本質的な解決 手段といえる。 In order to relax the hyperacute rejection reaction ^ transplantation between different buildings, c that is not considered conventionally to express the same H-type material and higher primates, such as humans in the donor, the conventional, natural antibodies Attention has been focused on the suppression of the capturer activation pathway after the binding of a protein to an antigen. Further, the present invention can be said to be a more essential solution to the conventional method in that the binding itself between the natural anti-G antibody and the G-type antigen in higher primates such as humans is suppressed.
第 I の発明は、 高等霊長類の F Tをコードする D N A配列を含む D N A棣 築物が導入され、 H型抗原を発現するように形質転換されていることを特徴とす る非笾長哺乳類の形質転換動物である。 この D N A構築物が導入された動物では、 F Tが発現され、 この F Tが N—ァセチルラク トサミ ンに作用して、 H型抗原を 発現する。 このため、 非 S長哺乳類に GT遣伝子があっても， FTが GTの基質 である N—ァセチルラク トサミ ンを消費すると、 内在される非霍長哺乳類の GT と N—ァセチルラク トサミ ンとの結合か抑制され、 G型抗原の発現が抑制される β これらの外来 DNAは、 ヘテロの状態で保持されていても、 ホモの状 で保持 されていてもかまわないが、 ホモの状想で保持されることが好ましい想様である。 A first aspect of the present invention relates to a non-human mammal having a DNA sequence containing a DNA sequence encoding FT of a higher primate, wherein the DNA construct has been transformed to express an H-type antigen. Transgenic animal. In the animal into which this DNA construct has been introduced, FT is expressed, and this FT acts on N-acetyl lactosamine to generate an H-type antigen. Express. For this reason, even if a non-S long mammal has a GT gene, when FT consumes N-acetyl lactosamine, which is a substrate of GT, the non-S long mammal's GT and N-acetyl lactosamine may be intermingled. bond or is inhibited, these foreign DNA beta expression is suppressed G group antigens, be held in a heterozygous state, but may be held at Jo homo-, held in homozygous Jo virtual It is a preferred idea to do so.
なお、 本発明において、 高等雷長類とは、 霊長類の真癀亜目のうち、 ヒ 卜 やチンパンジー、 オランウータン、 ヒヒ、 日本 ¾などが属する狭募猿類をいう。 また、 非高等霊長類とは、 霊長類の真擐亜目のうちの広鼻 ¾類及び原猿亜目をい う。 また、 非霊長哺乳類あるいは非霊長類哺乳動物とは、 霊長類でない哺乳類を いう。  In the present invention, higher primates refer to narrow monkeys belonging to humans, chimpanzees, orangutans, baboons, Japanese birds, etc. among primates. The non-higher primates are the broad-nosed primates and the protozoa among the primates. Non-primate mammals or non-primate mammals are mammals that are not primates.
また、 第 2の発明は、 高等签長類の FTをコードする DNA配列を含む第 1の DN A構築物と、 非霊長哺乳類の GT遺伝子に対してアンチセンスである D N A配列を含む第 2の DNA構築物とが導入され、 高等 長類の H型抗原を発現 する一方、 非霊長哺乳類の G型抗原の発現を低弒するように形踅転換されている ことを特攆とする非霊長哺乳類の形質転換動物である。  Also, the second invention comprises a first DNA construct comprising a DNA sequence encoding higher order FT and a second DNA comprising a DNA sequence which is antisense to the GT gene of a non-primate mammal. A non-primate mammal trait characterized in that it has been transformed to express a higher length H-type antigen while reducing expression of a non-primate mammal's G-type antigen. Convertible animal.
第 1の DNA構築物が導入されて形質転換されると、 FTが発現され， H型抗 原が発現される。 同時に G型抗原が低滅される。 また、 第 2の DNA構築物が導 入されると、 GTの発現が抑制されて、 G型抗原か低滅される。  When the first DNA construct is introduced and transformed, FT is expressed and H-type antigen is expressed. At the same time, type G antigens are reduced. In addition, when the second DNA construct is introduced, the expression of GT is suppressed, and the G-type antigen is reduced.
これらの外来 DNAは、 ヘテロの状態で保持されていても、 ホモの状態で保持 されていてもかまわないが、 ホモの伏態で保持されることが好ましい想様である c また、 第 Sの発明は、 非靈長哺乳類の GT¾伝子の一部に、 FTをコー ド する D N A配列を含む D N A榱築物が相同組換えにより挿入され、 非霊長哺乳類 の G型抗原を発現することなく高等霊長類の H型抗原を発現するように形質転換 されていることを特徴とする非 S長哺乳類の形質転換動物である。  These exogenous DNAs may be maintained in a heterologous state or a homologous state, but it is preferred that they be maintained in a homozygous state. The present invention relates to a method wherein a DNA construct containing a DNA sequence encoding FT is inserted by homologous recombination into a part of the GT gene of a non-primate mammal, thereby expressing a non-primate mammalian G-type antigen. A non-S long mammal transformed animal characterized by being transformed to express a primate H-type antigen.
この発明によると、 この DNA櫞築物により非 S長哺乳類の G T遣伝子に変異 が導入されることになるため、 GTは発現されず、 G型抗原は発現されない。 一 方、 同時に、 FTが発現され， この FTにより、 H型抗原が発現される。 また， この発明によれば， 非相同組換えにより生ずるとされる形質耘换動物への恶影 もない。 なお、 GTif伝子の一部において相同組換えを起こす DNA構築物は、 „ According to the present invention, a mutation is introduced into the GT gene of a non-S long mammal by this DNA construct, so that GT is not expressed and G-type antigen is not expressed. At the same time, FT is expressed, and H-type antigen is expressed by the FT. Further, according to the present invention, there is no effect on traited animals caused by heterologous recombination. DNA constructs that cause homologous recombination in part of the GTif gene are: „
96/28967 一 n 96/28967 one n
6一  Six
G丁遣伝子をコードする D N A配列と相同的な部分を有している。 It has a portion homologous to the DNA sequence encoding the G gene.
これらの外来 D N Aは、 ヘテロの状魅で保持されていても、 ホモの状態で保持 されていてもよいが、 ホモの状 ffiで保持されていることが好ましい »様である。 また、 第 4の発明は、 非霊長哺乳類の G T遣伝子の一部に、 F Tをコード する D N A配列を含む D N A構築物が相同組換えにより抻入され、 非 S長哺乳類 の G型抗原を発現することなく高等霊長類の H型抗原を発現するように形質転換 された非 S長哺乳類の形質転換動物から得られた移植用材料である。 この形質転 换動物から得られた細胞自体、 この細胞を構成要素とする組織、 «¾、 あるいは これら以外のその他の材料は、 幅広く高等霊長類への移植用材料に適用すること ができる。  These exogenous DNAs may be retained in a hetero state or in a homo state, but are preferably retained in a homo state ffi. In the fourth invention, a DNA construct containing a DNA sequence encoding FT is inserted into a part of the GT gene of a non-primate mammal by homologous recombination to express a G-type antigen of a non-S long mammal. This is a transplant material obtained from a non-S long mammal transformed animal that has been transformed to express a higher primate H-type antigen without any modification. The cell itself obtained from the transgenic animal, a tissue containing the cell as a component, ¾, or other materials can be widely used as a material for transplantation into a higher primate.
なお、 これらの発明における形質転換動物の好ましい態搛は、 前記非霊長 類哺乳動物が、 ブ夕であることである。 形質転換された動物がブタであると、 こ のブ夕によって高等霊長哺乳類への移植に適した材料を提供することができる。 さらに、 第 5の発明は、 非霊長哺乳類の G T遺伝子の一部に， F Tをコー ドする D N A配列を含む D N A構築物を相同組換えにより挿入し、 非靈長哺乳類 の G型抗原を発現することなく高等霊長類の H型抗原を発現するように形質転換 した非霊長哺乳類の形質転換動物の作出方法である。  In addition, a preferable aspect of the transgenic animal in these inventions is that the non-primate mammal is bush. If the transformed animal is a pig, this can provide material suitable for transplantation into higher primate mammals. Further, a fifth invention is to express a non-primate mammalian G-type antigen by inserting a DNA construct containing a DNA sequence encoding FT into a part of the GT gene of the non-primate mammal by homologous recombination. This is a method for producing a non-primate mammal transformed animal that expresses the H-type antigen of a higher primate.
この方法によると、 この D N A構 S物が導入されて形質転換されると、 G丁遠 伝子に変異が導入されて， G Tは発現されず， 代わりに F Tが発現され， H型抗 原が発現される。  According to this method, when this DNA construct is introduced and transformed, a mutation is introduced into the G protein and GT is not expressed, FT is expressed instead, and H-type antigen is expressed. Is expressed.
また. 第 6の発明は、 非 S長哺乳類の組織を高等霊長類に移植する隙の. 高等霊長類の自然抗体と非靈長哺乳類の抗原と（こよる拒絶反応を回避する方法て あって，  The sixth invention is directed to a method for avoiding rejection reaction between a natural antibody of a higher primate and an antigen of a non-primate mammal in a space for transplanting a tissue of a non-S long mammal into a higher primate. ,
非霊長哺乳類の G T逸伝子の一部に， F Tをコードする D N A配列を含む D N A構築物を相同組換えにより挿入して. 非霊長哺乳類の G型抗原を発現すること なく高等霊長類の H型抗原を発現するように形質転換した非 S長哺乳類を作出す る工程と. この工程により得られた前記非霊長哺乳類の祖維を高等霊長類に移植する工程 を含んだ方法である。 A DNA construct containing a DNA sequence encoding FT is inserted by homologous recombination into a part of the GT gene of a non-primate mammal. H-type of a higher primate without expressing the G-type antigen of the non-primate mammal Creating a non-S long mammal transformed to express the antigen. Transplanting the non-primate mammal progeny obtained in this step into a higher primate.
この発明によると， 非霊長哺乳類の組織においては， GSS [抗原でなく H型抗 原が発現されている。 このため， この組雄を高等靈長額に移植した際に， 高等霊 長類の有する抗 G自然抗体により拒絶されない。  According to this invention, non-primate mammal tissues express the HSS antigen but not the GSS [antigen]. Therefore, when this kumio is transplanted to the higher primate, it is not rejected by the anti-G natural antibody of the higher primate.
〔0面の 単な説明〕  [Simply explanation of surface 0]
図 1は、 前駆物質 （N—ァセチルラク トサミ ン） からの ABH型物質と G型物 質の合成柽路を示す図である。  FIG. 1 is a diagram showing a synthetic route of ABH-type substances and G-type substances from a precursor (N-acetyllactosamine).
図 2は、 実施例 Iの FT遣伝子を有するプラスミ ド pMAM/FTを構築操作 する過程図である。  FIG. 2 is a process diagram of the construction operation of the plasmid pMAM / FT having the FT gene of Example I.
図 3は、 実施例 Iの FT遺伝子専入用断片の模式図である。  FIG. 3 is a schematic view of the fragment for exclusive use of the FT gene of Example I.
図 4は、 実施例 2の pREP 8/AS/GTを構築操作する過程囟である。 る。  FIG. 4 is a process (2) of constructing and operating pREP8 / AS / GT of the second embodiment. You.
図 5は、 実施例 2の A S/G T¾伝子導入用断片の模式図である。  FIG. 5 is a schematic diagram of an AS / GT¾ gene introduction fragment of Example 2.
図 6は、 実施例 3の PREP 9/GT3- 4を構築操作する過程図である。 図 7は、 実施例 3の p RE P 9/GT 3— 厶 S s p Iノ厶 D r a I FIG. 6 is a process diagram of a construction operation of PREP 9 / GT3-4 of the third embodiment. FIG. 7 shows pREP9 / GT3-room SspI nom DraI of Example 3.
I Iを構集操作する過程及び ¾伝子断片 GT 3 - /FT/p 01 y Aの ¾製過 程を図示したものである。 This figure illustrates the process of assembling I and the process of manufacturing the gene fragment GT 3-/ FT / p 01 yA.
図 8は、 実施例 Sの pGT/FTを構築操作する過程図である。  FIG. 8 is a process diagram of a construction operation of the pGT / FT of the embodiment S.
図 9は、 5，C r リ リースアツセィの結果を示すグラフ図である。 FIG. 9 is a graph showing the results of 5 , Cr release atsee.
図 1 0は、 G丁追伝子のェクソン 2から 3の部分からフラグメント Aを得る方 法を示した図である。  FIG. 10 is a diagram showing a method for obtaining fragment A from exons 2 to 3 of G-choedogene.
図 1 1は GT¾伝子のェクソン 4から 6の部分からフラグメン ト Bを得る方法 を示した図である。  Figure 11 shows a method for obtaining fragment B from exons 4 to 6 of the GT¾ gene.
図 1 2は、 FT遣伝子のェクソン 1から 2から得られた c DNAを含んだブラ スミ ド Dを得る方法を示した図である。  FIG. 12 is a diagram showing a method for obtaining a plasmid D containing cDNA obtained from exons 1 and 2 of the FT gene.
図 1 Sは、 フラグメン ト Bとプラスミ ド Dからブラスミ ド Eを得る方法を示し た図である。  FIG. 1S is a diagram illustrating a method for obtaining a plasmid E from a fragment B and a plasmid D. FIG.
図 1 4は、 フラグメン ト Aとプラスミ ド Eからコンストラク トを得る方法を示 96/28967 N Figure 14 shows how to construct from fragment A and plasmid E. 96/28967 N
一 8一  One eight one
した図である。 FIG.
図 1 5は、 GT遣伝子とコンストラク 卜が同所性組換えを起こした場合の変異 遺伝子を示した図である。  FIG. 15 is a diagram showing a mutant gene when a GT gene and a construct undergo orthotopic recombination.
図 1 6において、 1 6 Aは FTをサザーンブロッテイング法により FT内配列 を検出した図であり、 1 6 Bは FT遣伝子の上流側の相同部分を P CRにより検 出した図であり、 1 6 Cは、 1 6 Cはコンストラク トの F丁の下流部分が相同組 換えを起こしていること P CRで確認したことを示す図であり、 1 6 Dはノザン プロッティ ングにより FT遣伝子の mRN Aを検出したことを示す図であり、 1 6 Eは L 9 2 9細胞が H抗原を発現していないことをレクチン UE A 1を用いた フ D—サイ トメ トリーを示した図であり、 1 6 Fは、 FTS伝子が導入された L 9 2 9細胞の H抗原をレクチン UE A 1で検出したことをフローサイ トメ トリ'一 で検出した図である。  In Fig. 16, 16A is a diagram in which the sequence in FT is detected from FT by Southern blotting, and 16B is a diagram in which the homologous portion of the FT gene on the upstream side is detected by PCR. Fig. 16C shows that 16C confirmed by PCR that homologous recombination occurred in the downstream part of the construct F-finger. 16D shows FT transmission by Northern plotting. Fig. 16 shows that mRNA of the offspring was detected. Fig. 16E shows that D9-29 cells do not express H antigen by D-cytometry using lectin UE A1. Fig. 16F is a diagram showing that the detection of the H antigen of L922 cells into which the FTS gene was introduced by the lectin UE A1 was detected by flow cytometry.
図 1 7において、 1 7 Aは、 FT遺伝子を P CRで検出した図であり、 1 7 B は、 コンスラク トの FTの上流側の相同部分において相同的組換えが行われたこ とを示す PC Rの図であり、 1 7 Cは、 コンストラク 卜の FTの下流側の相同部 分において相同的組換えが行われたことを示す PCRの図であり、 1 7 Dは FT S伝子の mRNAを検出した RT-P CRの図であり、 1 7 Eは L 9 2 9細胞が H抗原を発現していないことをレクチン UEA 1を用いて示したフローサイ トメ トリーの図であり、 1 7 Fは、 FT逸伝子か導入された L 9 2 9钿胞の H抗原を レクチン UEA 1で検出したことをフローサイ トメ トリーで示した図である。 In FIG. 17, 17A is a diagram in which the FT gene was detected by PCR, and 17B is a PC indicating that homologous recombination was performed in the homologous portion of the construct upstream of FT. R is a diagram, 17C is a PCR diagram showing that homologous recombination was performed in the homologous portion downstream of the FT of the construct, and 17D is an mRNA of the FTS gene. 17E is a flow cytometry diagram showing that L929 cells do not express H antigen using lectin UEA1, and FIG. FIG. 3 is a diagram showing, by flow cytometry, the detection of H antigen of L929 cells introduced with FT gene into the lectin UEA1.
〔発明を実施するための最良の形憨〕 [Best mode for carrying out the invention 憨]
FTを発現する形質転換動物  Transgenic animals expressing FT
ブタ等の非霊長類哺乳動物の脈管 ·血球系細胞において、 ヒ トなど高等霊 長類だけが持ち、 プタなどの非 S長類哺乳動物が持たない S伝子あるいはその発 現物質であるタンパク質を発現させるには、 形質転換動物を作成することが必要 になる。  In the vasculature and blood cells of non-primate mammals such as pigs, it is an S gene or its expression substance that only higher primates such as humans have and that non-S primate mammals such as septa do not. To express the protein, it is necessary to create transgenic animals.
ヒ トとヒ トとの移植における基本原則の一つとして血液型を合わせることが広 く認められているが、 いわゆる血液型すなわち A BO式の分類の基本物質は H型 物質であって、 それは脈管 '血球系細胞においては、 前述のように前 物質 （N ーァセチルラク トサミ ン） に梃転移醉素 FTが作用して形成される。 しかし、 ブ タなど非盪長類哺乳動物は、 FTでなく GTを持ち、 それによりヒ トとは異なつ た物質 （G型物質） を形成している。 It is widely accepted that blood group matching is one of the basic principles in human-to-human transplantation, but the basic substance of the so-called blood group, or ABO classification, is an H-type substance. In vascular blood cells, as described above, the previous substance (N Is formed by the action of FT on acetylsilactamine. However, non-chordate mammals such as pigeons have GT instead of FT, thereby forming a different substance (G-type substance) from human.
したがって、 例えば、 H型物質をその前駆物質から生成する糖転移酵素 FTを コー ドする FT遣伝子を含む外来适伝子断片をブタ等の受精卵に導入し、 この遣 伝子断片が染色体上に組み込まれた場合、 各紬胞でこの FT遺伝子か転写翻訳さ れ、 酵素 FTが產生されて H型物質を発現させることが期待できる。 Therefore, for example, an exogenous gene fragment containing an FT gene encoding glycosyltransferase FT, which produces an H-type substance from its precursor, is introduced into a fertilized egg such as a pig, and this gene fragment is transferred to the chromosome. When incorporated into the above, it is expected that each cell will be transcribed and translated from this FT gene, and the enzyme FT will be produced to express H-type substances.
H型物 Kを発現した非 II長哺乳類動物は、 組羝 ·血液の抗原型が高等 S長類 に近似され、 これらの動物の組維等を高等累長類に移植した際の超急性拒絶反応 を緩和することができ、 これらの動物の組維等は、 高等霊長類への移植に適した ものとなる。 Non-II long mammals expressing H-type K have similar acute serotypes in their blood and blood serotypes, and hyperacute rejection when transplanting the tissues of these animals into higher primates. The response can be mitigated, making these animal tissues suitable for transplantation into higher primates.
さらに、 Η型物質の場合には、 酵素 F Tが前駆物踅を消費すれば、 それだけ、 内在する酵素 G Tと前駆物質との結合が阻害され、 G型物質の生成が抑制される ことも期待できる。 なお、 F T¾伝子はプ夕染色体にくみこまれた後、 安定して 子孫に伝達されることが可能である。  Furthermore, in the case of type I substances, if the enzyme FT consumes the precursor 踅, it is expected that the binding of the endogenous enzyme GT to the precursor will be inhibited and the production of type G substances will be suppressed. . The FT¾ gene can be stably transmitted to progeny after it has been incorporated into the chromosome.
さらに、 本発明では、 ブタ等の非霊長類哺乳動物に内在する糖転移酵素、 例え ば、 G型物質をその前駆物質から生成する植転移酵素 G Tの逸伝子の発現を不活 性化する目的で、 G T¾伝子のアンチセンス D N Aを含む外来遣伝子断片をブ夕 受精卵に導入する。 この ¾伝子断片が、 僩体の染色体上に組み込まれた場合、 各 钿胞でこのァンチセンス D N Aが転写されれば、 その転写物は内在性の G T転写 産物と対合して、 G T¾伝子の翻訳が抑制されることが期待される。  Further, the present invention inactivates the expression of glycosyltransferases endogenous in non-primate mammals such as pigs, for example, the gene transfer of the plant transferase GT, which produces a G-type substance from its precursor. For this purpose, a foreign gene fragment containing the antisense DNA of the GT G gene is introduced into the fertilized egg. When this gene fragment is integrated on the human chromosome, if this antisense DNA is transcribed in each cell, the transcript will be paired with the endogenous GT transcript and the GT It is expected that translation of offspring will be suppressed.
この結果、 個体レベルでは、 酵素 F丁が積極的に発現されるとともに、 酵素 G Tの発現が抑制されて、 H型物質が生成される一方、 一暦 G型物質の生成が低滅 されたブ夕等を得ることができる。 なお、 アンチセンス D N A等の外来逭伝子断 片は、 染色体に組み込まれた後、 安定して子孫に伝達されることが可能である。 なお、 一旦、 染色体に外来遣伝子が組みこまれた後、 交配により外来适伝子に 閟してホモの状態にすることが可能であり、 高等霊長類の撺転移酵素を発現させ る外来适伝子に関してホモの状憨の系と、 非霊長哺乳類の糖転移酵素に関してホ モの状態の系とを交配させることにより、 非霍長哺乳類の酵素を発現するかわり に、 高等霊長類の糖転移酵素を発現して、 高等 長類の抗原型を呈した非霊長類 哺乳動物の系を得ることができる。  As a result, at the individual level, the enzyme F is actively expressed, the expression of the enzyme GT is suppressed, and H-type substances are produced, while production of G-type substances is reduced. Evening can be obtained. In addition, foreign gene fragments such as antisense DNA can be stably transmitted to progeny after being integrated into the chromosome. Once the foreign gene has been integrated into the chromosome, it can be homogenized by mating with the foreign gene, and the foreign gene that expresses the transferase of higher primates can be obtained. By mating a homozygous gene system with a homologous system for non-primate glycosyltransferases, instead of expressing non-primate mammalian enzymes, higher primate sugars can be used. A non-primate mammalian system that expresses a transferase and exhibits a higher serotype serotype can be obtained.
高等霊長類の糖転移酵素 Higher primate glycosyltransferases
本発明では、 高等霊長類として、 特にヒ トを対象することができる。  In the present invention, humans can be particularly targeted as higher primates.
高等 S長類の糠転移酵素とは、 高等 S長類において見いだされ、 非 S長啸乳類 の脈管 ·血球系細胞において見いだされない抗原の生成に閣与する糖転移酵素を いう。 具体例としては、 F Tを挙げることができる。 特に、 ヒ ト F Tは 〔E C 2 , 4. 1. 6 9〕 で特定される。 Higher S-long bran transferase refers to a glycosyltransferase that is found in higher S-longs and contributes to the production of antigens not found in non-S-long mammary vascular and blood cells. A specific example is FT. In particular, the human FT is [EC 2, 4.1.69].
高等 ¾長類の糖耘移酵素をコードする DN A配列を含む DN A桷篓物 DN A jujube containing a DNA sequence encoding a higher sugar-transferring enzyme
高等 長類の糠転移酵素をコードする DN A配列を含む DN A構築物は、 該糖転移酵素の遣伝子の DNA配列の他、 プロモータ， ターミネータ一等を有す ることができる。 SV 4 0の初期逭伝子のボリ A付加シグナルに挠くターミネ一 タ一は付加に適した配列の一つである。一股的にどの ¾伝子からとったボリ Aシ グナルかによつて遣伝子の発現のレベルはかなり左右されるが、 SV 4 0初期遗 伝子から取ったボリ A付加逭伝子が便れている。 このシグナルはターミネーター の前に置かれる。 FTを発現させることのできる外来遣伝子を形成するには、 少 なく とも FTの DNA配列を知る必要かある。 ヒ トの FTについては、 既に報告 されている （P r o c. t l . A c a d. S c i . USA. 87(1990), pp^67 4-6678) 。  A DNA construct containing a DNA sequence encoding a higher length bran transferase can have a promoter, a terminator and the like in addition to the DNA sequence of the gene for the glycosyltransferase. The terminator associated with the Boli A addition signal of the initial gene of SV40 is one of the sequences suitable for addition. Although the expression level of the transgene is considerably influenced by the B-A signal taken from which gene, the B-A addition from the initial SV40 gene is not I'm good. This signal is placed before the terminator. To form a foreign gene that can express FT, it is necessary to know at least the DNA sequence of FT. Human FT has already been reported (Proc. Tl. Acad. Sci. USA. 87 (1990), pp ^ 67 4-6678).
非霊長哺乳類の糖転移酵素 Glycosyltransferases from non-primate mammals
非笾長哺乳類として、 特に、 前記高等霊長類に対して異種間移植の対象と なる動物を対象とすることができる。 さらに、 家畜として飼育されるプタ、 ゥシ， ヒッジ、 ャギ等が好適である。 これらの家畜は、 飼育が容易で、 安定した供給が 可能なため、 移植用組雄の補充に適する。 さらに、 ブ夕か、 臓器の大きさや、 生 理学的 ·生化学的な観点、 発育が早く多産である点から好適である。  As the non-vertebrate mammal, in particular, an animal to be subjected to xenotransplantation to the higher primate can be targeted. In addition, stags, puppies, sheep, goats, etc. raised as domestic animals are suitable. These livestock are easy to breed and can be supplied stably, so they are suitable for replenishment of transplanting males. In addition, it is preferable from the viewpoint of organ size, physiological and biochemical viewpoints, and rapid growth and high productivity.
非霊長哺乳類の糖転移酵素とは、 非霊長哺乳類の脈管 ·血球系細胞において 見いだされ高等霊長類において見いだされない抗原の生成に関与する锭転移酵素 をいう。 具体例としては、 ブタゃマウスの GTを挙げることができる。 特に、 マ ウス GTは、 〔E C 2. 4. 1. 1 5 1〕 で特定される。  Glycosyltransferases in non-primate mammals are 锭 transferases involved in the production of antigens found in the vascular and blood cells of non-primate mammals and not found in higher primates. A specific example is the pig-to-mouse GT. In particular, the mouse GT is specified in [EC 2.4.1.15.1].
非霊長哺乳類の糖転移酵素を発現を抑制する DN A構築物 DNA constructs that suppress glycosyltransferase expression in non-primate mammals
非 長哺乳類の糖転移.酵素の発現を抑制する DN A構築物には、 該糖転移 酵素をコードする DN A配列に対してアンチセンスである DNA配列を含む DN A構築物、 あるいは、 該據転移酵素の ¾伝子において相同組換えされて、 該遣伝 子を他の遣伝子で置換したり、 該遣伝子に他の遺伝子を挿入したり、 変異を導入 することにより、 該 S伝子を破壊する DN A配列を含む DN A構築物を用いるこ とができる。 この DNA構築物を形成するには、 少なく とも非霊長哺乳類の糖転移酵素の遣 伝子配列を知る必要がある。 マウス GT遣伝子は、 既に報告されている （P r o c . Na t l . Ac a d. S c i. USA. （1989), pp.8227-8231)。 また、 ブ タ GTS伝子も Kに報告されている （Xe n o t r a n s p l a n t a t i onGlycosyltransferases of non-long mammals. DNA constructs that suppress the expression of enzymes include DNA constructs containing a DNA sequence that is antisense to the DNA sequence that encodes the glycosyltransferase, By homologous recombination in the gene, the gene is replaced by another gene, another gene is inserted into the gene, or a mutation is introduced into the S gene. A DNA construct containing a DNA sequence that disrupts DNA can be used. To form this DNA construct, it is necessary to know at least the non-primate glycosyltransferase gene sequence. The mouse GT gene has already been reported (Proc. Natl. Acad. Sci. USA. (1989), pp. 8227-8231). Also, the GTA gene of buta was reported to K (Xe notransplantation).
1 9 94 : 1 : pp.81-88) 。 1 994: 1: pp.81-88).
例えば、 GTのアンチセンス DNAを外来遣伝子とする場合には、 GT遣伝子 の c DN Aの一部又は全部をアンチセンス方向に組み込んだ DN A断片を DN A 構築物として形成する。  For example, when the antisense DNA of GT is used as a foreign gene, a DNA fragment in which a part or all of the cDNA of GT gene is incorporated in the antisense direction is formed as a DNA construct.
また、 相同組み換えによる場合には、 例えば GT遣伝子との相同領域を可及的 多く有するように形成するのが好ましい。  In the case of homologous recombination, for example, it is preferable to form a region having as many regions of homology with the GT gene as possible.
なお、 非靈長哺乳類の糖転移酵素の発現を抑制するには、 外来遣伝子の導入以 外に従来公知の方法によることもできる。  The expression of glycosyltransferase in non-primate mammals can be suppressed by a conventionally known method other than introduction of a foreign gene.
相同組換え用 DNA構築物 DNA construct for homologous recombination
また、 DNA構築物は、 染色体における非相同組換えのみならず、 相同組 換えを目的として構築することもできる。 この場合、 例えば、 ブタの GTit伝子 をターゲッ トに、 ヒ 卜の FT遣伝子を組み換えるようにすれば、 GTの発現の抑 制と、 FTの発現が同時に可能である。  In addition, the DNA construct can be constructed not only for non-homologous recombination on the chromosome but also for homologous recombination. In this case, for example, if the pig FT gene is recombined with the pig GTit gene as a target, the suppression of GT expression and the expression of FT can be simultaneously performed.
なお、 以上説明したような DN A構築物については、 遣伝子断片が染色体に組 み換えられた際に、 高率に前記糖転移酵素が発現される手段を用いることができ る。 例えば pMAMをベクタープラスミ ドとして外来逸伝子断片を調製した場合 には、 ステロイ ドの投与により高発現が誘導されうる。  For the DNA constructs as described above, a means for expressing the glycosyltransferase at a high rate when the gene fragment is recombined into a chromosome can be used. For example, when a foreign gene fragment is prepared using pMAM as a vector plasmid, high expression can be induced by administration of steroid.
DNA構築物の導入 Introduction of DNA construct
非霊長類哺乳動物への外来遣伝子の導入は、 レ トロウイルスを用いる方法 やマイクロインジ クションによる方法等多数あるが、 どの方法も採用すること ができる。 子孫を得るという点からは、 生殖細胞を形質転換することが望ましい t この場合の形質転換は、 非霊長類哺乳動物の胚钿胞や ES細胞あるいは、 受精卵 に対して行うのが望ましい。 There are many methods for introducing an exogenous gene into a non-primate mammal, such as a method using a retrovirus and a method using microinjection, and any method can be adopted. From the viewpoint of obtaining a progeny transformation when the t it is desired to germline transformation, non-primate mammalian embryo钿胞or ES cells, or, is preferably performed with respect to a fertilized egg.
形質転換動物 Transgenic animals
このように、 外来遗伝子が導入された非霊長類哺乳動物は、 種々の铀胞に 96/28967 一 1 3一 In this way, non-primate mammals into which foreign genes have been introduced can be transformed into various cells. 96/28967 1 1 3 1
おける高等霊長類の搪転移酵素の発現により、 あるいは同時に非霉長哺乳類の撺 転移酵素の発現の抑制により、 組繳 '血液の抗原型が高等霊長類に近似された動 物となる。 したがって、 これらの動物の組織等を高等 S長類へ移植した際の超急 性拒絶反応を锾和することができるようになり、 これらの動物の組織等は、 高等 霊長類への移植に適したものとなる。 また、 特に、 血管内皮細胞で発現された場 合には、 超急性拒艳反応が大きく锾和される。 さらに、 赤血球細胞における高等 霊長類の糖耘移酵素の発現により、 血液の抗原型が高等霊長類に近似された勅物 となり、 これらの動物の血液は高等 S長類の代替血液に適したものとなる。 また、 高等^長類の糖耘移酵素 F丁が導入された場合には、 非翥長哺乳類 の脈管 ·血球系細胞では、 H型物質か発現されると同時に G型物質が低铽される ことになり、 組織 '血液の抗原型か高等霊長類に近似された動物となる。 加えて、 同時に G Tの発現を抑制する外来遺伝子も導入された場合には、 より一層 G型物 質が低鉞され、 組織 ·血液の抗原型が極めて高等霉長類に近似された動物となり、 これらの組羝 ·血液は、 高等蓥長類への移植 ·代替物により適したものとなる。 特に、 F Tが血管内皮細胞表面に発現されれば、 異種間 K器あるいは組織移植に おける超急性拒铯反応が大きく緩和される。 また、 赤血球において発現されれば、 代替血液により適したものとなる。 Due to the expression of 搪 -transferase in higher primates, or at the same time, the expression of 転 移 -transferase in non-prolonged mammals is suppressed, the animal has a blood serotype similar to that of higher primates. Therefore, it is possible to mitigate the acute rejection reaction when these animal tissues are transplanted into higher S primates, and these animal tissues are suitable for transplantation into higher primates. It will be. In particular, when expressed in vascular endothelial cells, hyperacute rejection is greatly reduced. In addition, the expression of sugar priming enzymes in higher primates in red blood cells makes the serotype of blood similar to higher primates, and the blood of these animals is suitable as a substitute for higher S primates. Becomes In addition, when the high-length saccharifying enzyme F-fed is introduced, the H-type substance is expressed and the G-type substance is simultaneously reduced in the vascular and blood cell cells of non-long-term mammals. Tissues will be animals that are similar to blood serotypes or higher primates. In addition, when a foreign gene that suppresses the expression of GT is also introduced at the same time, the G-type substance is further reduced, and the animal becomes an animal whose tissue and blood serotypes are very similar to those of higher vertebrates. These knots and blood are more suitable for transplantation to higher limbs and alternatives. In particular, when FT is expressed on the surface of vascular endothelial cells, hyperacute rejection in xenogeneic organs or tissue transplantation is greatly reduced. Also, if expressed in erythrocytes, it will be more suitable for blood substitutes.
また、 相同組換えにより、 G Tのかわりに F Tが発現するように形質転換 された形質転換動物においては、 染色体上の 本以上の G T¾伝子座のうち、 一 本か存在せず、 同時に F Tが少なく とも 1本存在するようになるため、 交配によ り染色体上に G Tが 1本も存在せず、 その代わりに F Tか 2本存在する個体を得 ることができる。 こう した個体はドナーとして、 レシピエントの A B O式の血液 型のいかんをとわずに、 ヒ ト等の移植に適した移植材料を提供する。  In the transgenic animal transformed by homologous recombination to express FT instead of GT, one or more of the GT G gene loci on the chromosome do not exist, and FT As a result, at least one GT exists on the chromosome, so that an individual with FT or two GTs can be obtained instead. Such an individual provides a transplant material suitable for transplantation of a human or the like as a donor, regardless of the recipient's ABO blood type.
產業上の利用可能性 上 の Business availability
このように、 かかる形質転換された非霊長類哺乳動物は、 ヒ ト等の高等霊 長類に移植の可能な組織 '血液を有するため、 ヒ ト等の組織等の代替物の提供源 となる。 また、 これらの動物が安定して飼育されることにより、 必要時に、 必要 な組雉等を提供可能となり、 移植組維等の貯蔵体となる。 96/28967 , t Thus, such a transformed non-primate mammal has a tissue that can be transplanted into a higher primate such as a human, and thus becomes a source of a substitute for a tissue such as a human because it has blood. . In addition, if these animals are stably bred, they will be able to provide necessary pheasants and the like when needed, and will be used as storage for transplanted tissues. 96/28967, t
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また、 非 S長類哺乳動物における高等 S長類特有の FTS伝子の発現によ り、 異種 ra移植の場合に超急性拒絶反応を緩和できる動物や材料を提供する方法 は、 従来にない新規なものであるとともに、 異種間移植における超急性拒絶反応 の接和のために、 より本質的な解决をもたらすものである。 In addition, a method to provide animals and materials that can mitigate hyperacute rejection in the case of xenograft ra transplantation by expressing the FTS gene specific to higher S-length mammals in non-S-length mammals is an unprecedented new method. And provide a more essential solution for the reconciliation of hyperacute rejection in xenotransplantation.
本発明の非靈長哺乳類の形質転換動物によれば、 その組織 ·血液において、 高等霊長類の糖鎖抗原が生成されるため、 あるいは同時に非霊長哺乳類の糖鎖抗 原の生成が抑制されるため、 非 s長類哺乳動物の組織を異種間移植となる高等靈 長類に移植した場合に超急性拒絶反応を钹和することができ、 従来の人工血管や 人工臓器、 あるいは同種間で補充される瞜器、 組磁等に代わり得る材料を提供す ることができる。  According to the transgenic non-primate mammal of the present invention, a sugar chain antigen of a higher primate is produced in its tissue and blood, or simultaneously, the production of a sugar chain antigen of a non-primate mammal is suppressed. As a result, hyperacute rejection can be mitigated when non-s long mammal tissues are transplanted into higher primates, which are allogeneic transplants, and can be replaced with conventional artificial blood vessels, artificial organs, or allogeneic transplants. It is possible to provide a material that can be used in place of a magnetic device, a magnet assembly, and the like.
なお、 本発明により非蓳長類哺乳動物を形質転換することは、 これらの'動 物にいかなる被害あるいは***を与えることがなく、 また、 本発明は、 形質転換 された非 ϋ長類哺乳動物の生存、 生殖等に恶彩饗を及ぼすものではない。 さらに、 本発明は、 ヒ トをはじめ他のいかなる動物に対しても悪影 gを及ぼすものではな い。 以下に本発明の実施例を示す。 本発明に於ける外来 s伝子に関して、 既に述べ た 2種類の搪転移酵素、 即ち G Tit伝子と F T遺伝子の c D N Aを例示できる。 ただし、 以下の実施例は、 請求の IS囲を制限するものではない。  In addition, transforming a non-mammal mammal according to the present invention does not cause any damage or abuse to these animals. It does not affect the survival, reproduction, etc. of the fish. Further, the present invention does not exert any adverse effects on humans or any other animals. Hereinafter, examples of the present invention will be described. Regarding the exogenous s gene in the present invention, the two types of 搪 -transferases already described, namely the G Tit gene and the cDNA of the F T gene, can be exemplified. However, the following embodiment does not limit the IS area of the claim.
(実施例 1 ) (Example 1)
導入用 ¾伝子を含むブラスミ ド （PMAMZFT) の構築及びその ¾伝子導入 用断片の調製  Construction of a plasmid (PMAMZFT) containing a gene for introduction and preparation of a fragment for introduction of the gene
( 1 ) P CR法による FT遣伝子断片の調製  (1) Preparation of FT gene fragment by PCR method
P r 0 c . Na t l . Ac a d. S c i . USA, 87(1990). pp6674_6678に報 告されたヒ 卜の FT 〔EC 2. 4. 1. 96〕 の DNA配列に基づいて、 そのス 夕一トコ ドンとストップコ ドンを含むような形で PC R用の 2種類のブライマー を合成した。 なお、 その 5' 側のプライマーには、 制限酵素 Nhelのサイ トを付け た、 また 3' 側のプライマーには、 制限酵素 Xho【のサイ トを付けた。 一方、 健康な成人の血液より白血球を採取し全 RNAを抽出し、 逆転写酵素を 用いて、 全 RNAに対応する c DNAを合成した。 の c DNAを鎵型にして、 前記のプライマーを用いて P CRを施行した。 USA, 87 (1990) .pp6674_6678, based on the DNA sequence of human FT [EC 2.4.1.96]. Two types of primers for PCR were synthesized in such a way that they contained both a tocodon and a stop codon. The 5 'primer was provided with a restriction enzyme Nhel site, and the 3' primer was provided with a restriction enzyme Xho [site. On the other hand, leukocytes were collected from healthy adult blood, total RNA was extracted, and cDNA corresponding to total RNA was synthesized using reverse transcriptase. The cDNA was subjected to PCR using the above primers.
本 PCR施行により得られた c DNA断片を 1 ¾ァガロースゲル 気泳動にか けたところ、 約 llOObpの長さのバン ドを得た。 この PCR産物を p GEM— T (Promega 社製） に挿入した上で自動塩基配列読み取り機で塩基配列を読み取つ たところ、 前記既報の通りの FT遣伝子の塩基配列を有していた。 そこでこの c DNAを 0. 89£Low Melting Point (以下、 L M Pという。 ） ゲルで電気泳動 して精製した後、 制限酵素 Nhelと Xholで処理し、 レジンを用いた Promega 社の Wi zardDNA精製システム （以下、 Wizardシステムという。 ） により精製 .分離し た。 なお、 以下各種 DNA断片の精製分離は、 LMPゲル電気泳動と Wizardジス テムとを用いることにより行った。  When the cDNA fragment obtained by this PCR was subjected to 1-agarose gel electrophoresis, a band having a length of about llOObp was obtained. After inserting this PCR product into pGEM-T (Promega) and reading the base sequence with an automatic base sequence reader, it had the base sequence of the FT gene as previously reported. Therefore, this cDNA was purified by electrophoresis on a 0.89 £ Low Melting Point (hereinafter, LMP) gel, treated with restriction enzymes Nhel and Xhol, and Promega's Wizard DNA purification system using resin ( Hereinafter, it is referred to as Wizard system. In the following, purification and separation of various DNA fragments were performed by using LMP gel electrophoresis and Wizard system.
(2) pMAMZFTの構築 (2) Construction of pMAMZFT
哺乳類発現ベクターの一種である pMAM (CL0NETECH 社製、 Lee.F. (1981) N ature 294:228)のマルチクローニング部位 （MC S) を制限酵素 Nhelと Xholで処 理し、 この断片を LMPゲル ¾気泳動及び Wizardシステムで精製 .分雌した。 こ のベクターに精製 '分離した FT遗伝子の c DN Aを、 T4 DNAリガーゼによ り挿入させた。 このプラスミ ド DN Aによって、 コンビテント状想の JM 1 0 9 細菌細胞を形質転換させて、 得られた形質転換体をアンビシリ ンプレートで選択 した。 個々のコロニー毎に L B培地 （組成： Bacto-tryprone 10g(DIFC0社製） 、 Bacto-yeast extract 5g(DIFC0社製) 、 NaCl 10gを水にて 1Lとする、 以下同じ。 ) で增链させ、 ミニブラスミ ド調製を行った。 抽出したプラスミ ド DNAを種々 の制限酵素を使って消化した後に電気泳動して、 挿入断片のサイズ及び位置方向 を斑べた。 その結果、 正しい方向に組み込まれているプラスミ ドを、 pMAMZ FTと称した （02参照） 。  The multicloning site (MCS) of pMAM (a product of CL0NETECH, Lee.F. (1981) Nature 294: 228), a type of mammalian expression vector, is treated with restriction enzymes Nhel and Xhol, and this fragment is subjected to LMP gel. Purified by electrophoresis and Wizard system. The purified FT gene cDNA was inserted into this vector using T4 DNA ligase. The plasmid DNA was used to transform JM109 bacterial cells in a combinatorial concept, and the resulting transformants were selected on an Ambicillin plate. Each individual colony was allowed to grow with an LB medium (composition: Bacto-tryprone 10 g (manufactured by DIFC0), Bacto-yeast extract 5 g (manufactured by DIFC0), and NaCl 10 g to make 1 L with water, the same applies hereinafter). A mini-plasmid was prepared. The extracted plasmid DNA was digested with various restriction enzymes and subjected to electrophoresis to spot the size and position of the inserted fragment. As a result, the plasmid integrated in the correct direction was called pMAMZ FT (see 02).
( 3) 遗伝子 «入用断片の調製 (3) 遗 Men 子 «Preparation of input fragments
さらに、 逭伝子導入のための直鑌状断片とするために、 大腸菌 JM 1 0 9/p MAM/FT株を LB培地で大量培養した後に、 プラスミ ド調製を行った。 さら に、 得られたプラスミ ドを C s C 1密度勾配遠心法により楕製した。 棺製したブ ラスミ ド 1 0 gを、 制限酵素 P V u 】 と B amH Iで消化後、 この断片を精製 -分雠し、 FTS伝子導入用断片とした。 この断片の構築模式図を図 3に示す。 Furthermore, E. coli JM109 / p was used to obtain a linear fragment for gene transfer. Plasmid preparation was performed after mass-culturing the MAM / FT strain in LB medium. Further, the obtained plasmid was oval by CsC1 density gradient centrifugation. After digestion of 10 g of the coffin-produced brasmid with the restriction enzymes PVu] and BamHI, this fragment was purified and separated to obtain a fragment for FTS gene transfer. A schematic diagram of the construction of this fragment is shown in FIG.
(実施例 2 ) (Example 2)
導入用遗伝子を含むブラスミ ド （PRE P 8/AS/GT) の構築及びその導 入用遗伝子断片の調製  Construction of a plasmid (PRE P 8 / AS / GT) containing a gene for introduction and preparation of a fragment of the gene for introduction
( 1 ) アンチセンス GTの調製  (1) Preparation of antisense GT
既報 (X e n o t r a n s p l a n t a t i o n 1 9 9 4 ; 1 ： ρρ 8 1 - 8 8 ) に報告されたブタ GT遣伝子の DNA配列に基づいて、 そのスタートコ ドン とストップコ ドンを含むような形で 2種類の PC R用のプライマーを合成した。 なお、 その 5 ' 側のブライマーには、 制限酵素 Xholのサイ トを付けた、 また 3 ' 側のブライマーには制限酵素 Nhdのサイ 卜を付けた。  Based on the DNA sequence of the porcine GT gene reported in the previous report (Xenotransplantation 1994; 1: ρρ81-88), two types of DNA, including their start and stop codons, were used. Primers for PCR were synthesized. The 5'-side primer was provided with a restriction enzyme Xhol site, and the 3'-side primer was provided with a restriction enzyme Nhd site.
—方、 健康なブタの血液より白血球を採取し、 その全 RNAを抽出し、 逆転写 酵素を用いて全 RN Aに対応する c DNAを合成した。 この c DN Aを铸型にし て、 前記のプライマーを用いて P CRを施行した。  On the other hand, leukocytes were collected from blood of healthy pigs, the total RNA was extracted, and cDNA corresponding to total RNA was synthesized using reverse transcriptase. This cDNA was transformed into type III, and PCR was performed using the primers described above.
本 P CR施行により得られた c DNA断片を 1 %ァガロースゲル菴気泳動にか けたところ、 約 llOObpの長さのバンドを得た。 この P CR¾物を p GEM— T (Proraega 社製） に挿入した上で、 自動塩基配列読み取り摟で塩基配列を読み取 つたところ、 前記既報の通りの GT遣伝子の塩基配列を有していた。 そこでこの c D N Aを L M Pゲルで電気泳動して精製した後、 制限酵素 Nhelと Xholで処理し、 Wizardシステムを用いて情製 ·分雜した。  When the cDNA fragment obtained by this PCR was subjected to 1% agarose gel electrophoresis, a band having a length of about llOObp was obtained. After inserting this PCR product into pGEM-T (proraega), the base sequence was read by automatic base sequence reading (1), and it had the base sequence of the GT gene as previously reported. . Therefore, the cDNA was purified by electrophoresis on an LMP gel, treated with restriction enzymes Nhel and Xhol, and then separated and distributed using a Wizard system.
( 2 ) R E P 8 /A S/GTの構築 (2) Construction of REP 8 / A S / GT
哺乳類発現ベクターの一種である P R E P 8 (Invitorogen 社製、 George. R.. et al. Gene.81.285(1989) ) の MC Sを制限酵素 Xholと Nhelで処理し、 精製 ·分 離した。 このベクターに、 T 4 DNAリガーゼにより、 精 ¾ ·分離した GT遣伝 子の cDNAを、 揷入した。 このプラスミ ド DN Aによって、 コンビテン ト伏態 の JM 1 09細菌細胞を形質転換させて、 得られた形質転換体をアンピシリ ンブ レートで選択した。 個々のコロニー毎に L B培地で増殖させ、 ミニブラスミ ド調 製を行った。 抽出したプラスミ ド DNAを種々の制限酵素を使って消化した後に 電気泳動して、 挿入断片のサイズ及び位置方向を調べた。 その锫果、 正しく アン チセンス方向に組み込まれているプラスミ ドを、 PREP 8/AS/GTと称し た （図 4参照） 。 MCS of PREP 8 (Invitorogen, George. R. et al. Gene. 81.285 (1989)), a kind of mammalian expression vector, was treated with restriction enzymes Xhol and Nhel, purified and separated. This vector is used to transfer the purified and isolated GT gene by T4 DNA ligase. Offspring cDNA was introduced. The plasmid JM109 bacterial cells were transformed with the plasmid DNA and the resulting transformants were selected with ampicillin plate. Each individual colony was grown in LB medium and mini-plasmid preparation was performed. The extracted plasmid DNA was digested with various restriction enzymes and subjected to electrophoresis to examine the size and the orientation of the inserted fragment. As a result, the plasmid correctly incorporated in the antisense direction was called PREP 8 / AS / GT (see Fig. 4).
(3) 導入用退伝子断片の調製 (3) Preparation of gene fragments for introduction
さらに、 遺伝子導入のための直鎖状断片とするために、 大腸菌 JM1 0 9/p REP 8ZAS/GT株を L B培地で大量培養した後に、 プラスミ ド調製を行つ た。 さらに、 得られたブラスミ ドを C s C 1密度勾配遠心法により精製した。 精 製したブラスミ ド 1 0 gを、 制限酵素 Xb a Iと P s t Iで消化後、 この断片 を LMPゲル ¾気泳動及び Wizardシステムで精製 ·分雠し、 AS/GT遺伝子導 入用断片とした。 この断片の構築椟式図を図 5に示す。  Furthermore, Escherichia coli JM109 / pREP8ZAS / GT strain was mass-cultured in LB medium to prepare a linear fragment for gene introduction, and then plasmid was prepared. Further, the obtained plasmid was purified by CsC1 density gradient centrifugation. After digesting 10 g of the purified plasmid with the restriction enzymes XbaI and PstI, this fragment was purified and separated by LMP gel electrophoresis and the Wizard system, and then used as a fragment for AS / GT gene transfer. did. The construction scheme of this fragment is shown in FIG.
(実施例 3) (Example 3)
相同組換え用适伝子を含むプラスミ （pREP 9ZGTZFT) の構築及び 導入用遣伝子断片 （P G丁/ FT) の調製  Construction of plasmid containing plasmid for homologous recombination (pREP 9ZGTZFT) and preparation of gene fragment for transfection (PG-cho / FT)
以下、 マウスの例を示すかブタにおいても基本的な考え方は同じである。  Hereinafter, the basic concept is the same for a mouse or a pig.
( 1 ) p RE P 9/GT 3 - 4の構築  (1) Construction of pREP 9 / GT 3-4
以下、 pREP 9ZGT3— 4の桷築を図 6に基づいて説明する。  Hereinafter, the construction of pREP 9ZGT3-4 will be described with reference to FIG.
既報 （P r o c. a t l . Ac d. S c i . USA, 86(1989), pp.8227-8 231)のマウスの GT 〔E C 2. 4. 1. 1 5 1〕 の配列に基づいてェキソン 3か ら 4までの部分を P CR法にて抽出するために、 ェキソン 3内のセンスブライマ 一として制限酵素 Kpn Iの切断部位をつけたブライマー （ρ/Αとする） 、 又 アンチセンスプライマ—としてェキソン 4内のスター トコ ドンの部位に制限酵素 S s ρ Iの切断部位を含み、 その下流に制限酵素 H i n d I I Iの切断部位をつ けたブライマー （pZB) を合成した。 これらのプライマー p/A及び p/Bを用いて P CR法を施行し、 得られた断 片を、 制限酵素 Kpn I及び H i n d I I Iで切断した後、 精製 '分離し、 これ を GT 3 - 4と称した。 USA, 86 (1989), pp. 8227-8231, based on the sequence of the mouse GT [EC 2.4.1.1.151] exon. In order to extract parts 3 to 4 by the PCR method, a primer with a restriction enzyme Kpn I cleavage site (referred to as ρ / Α) as a sense primer in exon 3 and an antisense primer A primer (pZB) containing a restriction enzyme SsρI cleavage site at the start codon site in exon 4 and a restriction enzyme HindIII cleavage site downstream thereof was synthesized. The PCR method was performed using these primers p / A and p / B, and the obtained fragment was cut with restriction enzymes KpnI and HindIII, purified and separated, and this was separated into GT3- Called 4.
—方、 発現ベクターである pREP 9 Unvitrogen社） を制限酵素 BamH I で切断し、 T4DNAボリ メラ -ゼによりその断端を平滑にし、 T4DNAリガ ーゼにより self - ligationし、 制限酵素 B a mH Iの切断部位が欠失した発現べ ク夕ーを、 pREP 9/厶 B amH Iと称した。 On the other hand, the expression vector (pREP 9 Unvitrogen) is cut with the restriction enzyme BamHI, the end is blunted with T4 DNA polymerase, self-ligated with T4 DNA ligase, and the restriction enzyme BamHI is used. The expression vector from which the cleavage site was deleted was designated as pREP9 / amBamHI.
これを更に制限酵素 D r a I I Iで切断し、 T 4 D N Aボリ メラ-ゼによりそ の断端を平滑にし、 T 4 DNAリガーゼにより seli-ligationし、 制限酵素 D r a I I Iの切断部位か欠失した発現ベクターを、 pREP 9ZABamH Iノ厶 Dr a I I Iと称した。 このベクターを制限酵素 K p n I及び H i n d I I Iで 切断し、 上記 GT 3— 4を挿入したブラスミ ドを PRE P 9ZGT 3— 4と称し た。  This was further digested with the restriction enzyme DraIII, the end was blunted with T4 DNA polymerase, and seli-ligated with T4 DNA ligase to delete the restriction enzyme DraIII cleavage site. The expression vector was called pREP 9ZABamH I Nom Dr a III. This vector was digested with restriction enzymes KpnI and HindIII, and the plasmid into which the above GT3-4 was inserted was designated as PREP9ZGT3-4.
(2) pREP 9/G丁 3— 4/FT/AS s p I /AD r a 】 I Iの構築 以下、 pR E P 9/GT 3 4 FT/厶 S s p I Z厶 D r a I I Iの桷築 を図 7に基づいて説明する。  (2) Construction of pREP 9 / G cho 3-4 / FT / AS sp I / AD ra】 II The following figure shows the construction of pREP 9 / GT 34 FT / m S sp IZ m Dra III. It will be described based on the following.
ヒ ト FT (P r o c. N t l. Ac a d. S c i . USA, 87(1990). pp667 4-6678) の配列に基づき、 そのスター トコ ドンとストップコ ドンを含む発現部位 を P CR法にて抽出するために、 スター トコ ドンを含む 5' 側のセンスプライマ 一 (p/C) には制限酵素 Nh e Iと Dr a I I I切断部位を、 この頓で付けた t —方、 FTのストップコ ドンを含むアンチセンスプライマー （pZD) にはそ の 3' 側に制限酵素 Xh 0 I切断部位を付けた。 これらのプライマーを用いて P CR法を施行し、 得られた断片を制限酵素 Nh e I及び X ho Iで切断し、 pR E P 9/GT 3— 4を制限酵素 Nh e I及び Xh o Iで切断したプラスミ ドに揷 入する。 これを p R E P 9ZGT 3— 4ZFTと称した。 このプラスミ ド PREP/GT3 - 4ZFTを制限酵素 S s p I及び D r a I I Iで切断すると平滑末端が生成された。 ここで T 4 DNAリガーゼにて処理すると sdf— ligationにより両者末端が连 結し、 GTの発現開始部位に FT c DNAの発現開始部位が位置された。 USA, 87 (1990) .pp667 4-6678), and the expression site including its start codon and stop codon was replaced with the PCR fragment based on the sequence of human FT (Proc. Ntl. Acad. Sci. to extract in law, t restriction enzyme Nh e I and Dr a III cleavage site on the sense primer one (p / C) of 5 'side including the star Toco Don, gave at the needed basis - how, FT An antisense primer (pZD) containing this stop codon was provided with a restriction enzyme XhoI cleavage site on its 3 'side. The PCR method was performed using these primers, the resulting fragment was digested with restriction enzymes NheI and XhoI, and pREP9 / GT3-4 was digested with restriction enzymes NheI and XhoI. Insert the cut plasmid. This was called pREP 9ZGT 3-4ZFT. When this plasmid PREP / GT3-4ZFT was cleaved with restriction enzymes SspI and DraIII, blunt ends were generated. Here, when treated with T4 DNA ligase, both ends were linked by sdf-ligation, and the FT cDNA expression initiation site was located at the GT expression initiation site.
このブラスミ ドを p R E P 9/GT 3— 4/?丁/厶3 s p I /ΔΌ r a i l Iと称した。 以上に於いてこれら制限酵素 K pn H i nd l ! S s p I, Nh e 】、 Dr a l I I、 及び Xh o Iがいずれもェキソン 3からェキソン 4ま での断片及び FT c DN Aの発現部位の断片を切断しないことが既に確かめられ ている。  This brassmid was designated as pREP9 / GT3-4 /? C / m3spI / ΔΌrailI. In the above, these restriction enzymes KpnHindl! SspI, Nhe], DralII, and XhoI were all fragments from exon 3 to exon 4 and the expression site of FTcDNA. It has already been found not to cut fragments.
(3)導入用遗伝子断片 pGT/FTの調製 (3) Preparation of gene fragment pGT / FT for introduction
以下、 p GT/ FTの調製について図 7及び図 8に基づいて説明する。  Hereinafter, the preparation of pGT / FT will be described with reference to FIGS. 7 and 8.
PRE P 9の p 01 y Aの 3' 側を含みかつ制限酵素 B a m H I切断部位を'含 む形でアンチセンスプライマ— （p/E) を合成し、 これとセンスプライマー (P/A) とを用いて、 pREP 9/GT3- 4/FT/AS s p I /AD r a An antisense primer (p / E) was synthesized containing the 3 'side of p01yA of PRE P9 and containing the restriction enzyme BamHI cleavage site, and a sense primer (P / A) And pREP 9 / GT3-4 / FT / AS sp I / AD ra
I I Iに対し、 PCR法を施行し、 得られた断片を制限酵素 Kp n I及び BamPCR was performed on I I I, and the obtained fragment was digested with restriction enzymes Kpn I and Bam
H Iで切断して精製 ·分雌し、 得られた断片を GT 3 - 4/FTZp o l yAと 称した （図 7参照） 。 The fragment was purified by cutting with HI and separated, and the obtained fragment was designated as GT3-4 / FTZpolyA (see FIG. 7).
—方、 発現べクタ一 p R E P 9を制限酵素 K p n Iと B amH Iで切断し、 上 記の断片 GT 3 - 4/FT/p 01 y Aを揷入した。 これを pREP 9ZGT3 On the other hand, the expression vector pREP9 was digested with restriction enzymes KpnI and BamHI, and the above fragment GT3-4 / FT / p01yA was introduced. This is pREP 9ZGT3
- /FT/P 01 y Aと称した。 -Called / FT / P 01 y A.
GTのェキソン 4からェキソン 6までの部分を、 センスプライマー （pZF) ， アンチセンスブライマ- (p/G) とも制限酵素 B amH Iの切断部位を付け、 PCR法にて抽出し、 この断片を制限酵素 B amH Iで切断し、 得られた断片を、 制限酵素 B amH Iで開列したプラスミ ド p R E P 9 /GT 3— 4ノ F TZp o 1 y Aに挿入した （B amH Iが、 GT 3 - 4、 FT、 p o 1 y A及び T 4一 7 を切断しないことは既に確かめられている） 。 The exon 4 to exon 6 part of the GT was digested with the restriction enzyme BamHI site for both the sense primer (pZF) and antisense primer (p / G), and extracted by PCR. The fragment obtained by digestion with the restriction enzyme BamHI was inserted into a plasmid pREP9 / GT3—4noFTZpo1yA that had been opened with the restriction enzyme BamHI (BamHI was GT It has already been ascertained that 3-4, FT, po 1 y A and T 4-7 do not cut).
得られたプラスミ ドを PREP 9ZGT3— 4/FTZp o l yAZGT4— 6、 或いは簡単に、 pREP 9/GTZFTと称した。 このブラスミ ドに対し、 プライマー p/A及び p/Gを用いて PCR法を施行 した。 精製後得られた断片を受精卵への導入用断片とし、 GT 3— 4/FT/p 0 1 y A/GT 4一 6、 或いは簡単に p G TZF Tと称した。 このようにして作成した導入用遣伝子断片は、 GTの染色体の遣伝子と相同性 が高く、 相同組み換えを起こすことが期待される。 相同組み換えを起こした場合 は、 GTの代わりに F丁が発現するのでその個体 （F o) は GTも FTも発現す る。 しかし交配を 1、 2世代行うと、 メ ンデルの法則により、 FTのみを持った 価体を得ることが出来る。 この偭体こそが、 非霊長類哺乳動物に特有な抗原を持 たずに高等靈長類特有の抗原を持つに至った、 形質転換動物である。 マウスの G Tとブ夕の GTとではホモロジ一が高いのでマウスの GTに基づいた相同組み '换 え遣伝子をブタに適用することができる。 The resulting plasmid was designated as PREP 9ZGT3-4 / FTZpolyolAZGT4-6, or simply as pREP9 / GTZFT. The plasmid was subjected to PCR using primers p / A and p / G. The fragment obtained after purification was used as a fragment for introduction into a fertilized egg, and was referred to as GT3-4 / FT / p01yA / GT416, or simply pGTZFT. The transgene fragment for introduction thus prepared has high homology with the gene of the GT chromosome and is expected to undergo homologous recombination. When homologous recombination occurs, the individual (F o) expresses both GT and FT because F-cho is expressed instead of GT. However, after one or two generations of crossing, a valence having only FT can be obtained according to Mendel's law. This is the transgenic animal that has no antigen specific to non-primate mammals but has an antigen specific to higher primates. Since the homology between mouse GT and Busu GT is high, the homologous recombination gene based on mouse GT can be applied to pigs.
(実施例 4) (Example 4)
導入用遺伝子断片 （PMAM/FT及び p RE P 8/AS/GT) のブ夕受精 卵への注入及びその受精卵の移植  Injection of transgene fragments (PMAM / FT and pREP8 / AS / GT) into fertilized eggs and transplantation of the fertilized eggs
( 1 ) 導入用遣伝子断片 （pMAMZFT及び p RE P 8/GT) のプタ受精卵 への注入  (1) Injection of transfection gene fragments (pMAMZFT and pREP8 / GT) into fertilized egg
実驗動物として家畜豚を用いた。 受精卵提供用のブ夕としてし andraceと White Yorkshire の交配豚で、 生後約 6ヶ月の、 まだ自然発情を来す前の雌ブタを用い た。 この雌ブタに***誘発剤- pMS (妊馬血清、 シグマ社製、 G4877)及び h CG (ヒ ト絨毛ゴナ ドトロピン、 シグマ社製、 CG5)を注射し、 Duroc 系の成熟雄ブタ から採取した铕液により人工受精を行い、 卵管を M 2培地で灌流することにより 受堉卵を採取した。 これらの受精卵の前核は脂肪滴に Sわれているために直視出 来ないため、 受精卵を lOOOOgで 1 0分間遠心することにより、 脂肪滴と前核を分 離し、 微分干渉顕微镔で直視下においた。 受楕卵への遣伝子の注入に於いては、 マウス受精卵に於ける遣伝子導入の既報 ( Hogan. et al. , In manipulating the Mouse Embryo.. Cold Spring Harbor 96/28967 - 2 \ Domestic pigs were used as experimental animals. A sow of andrace and White Yorkshire, a sow of about 6 months of age and not yet in natural estrus, was used as a bush for providing fertilized eggs. This sow was injected with an ovulation-inducing agent-pMS (pregnant horse serum, Sigma, G4877) and hCG (human chorionic gonadotropin, Sigma, CG5), and collected from a mature Duroc strain bovine. Artificial fertilization was performed using the solution, and the fertilized eggs were collected by perfusing the oviduct with M2 medium. Since the pronucleus of these fertilized eggs cannot be seen directly because they are covered by lipid droplets, centrifuging the fertilized eggs at lOOOOg for 10 minutes separates the lipid droplets from the pronucleus, and differentiates them by differential interference microscopy. It was under direct vision. Regarding the injection of gene into oval eggs, a report on the introduction of gene into mouse fertilized eggs (Hogan. Et al., In manipulating the Mouse Embryo .. Cold Spring Harbor 96/28967-2 \
Laboratory Press. 1986、 Pinkert. et al.. Transgenic animal, technology.. Ac ademic press . Inc.1994 )に記載されているマイクロインジ クション法を、 ブ 夕受精卵に適用した。 即ち、 培養液中で受精卵を保持用ガラスビぺッ トで固定後， 微小ガラスピぺッ トを用いて導入用 δ伝子断片の溶液 2 1を雄性前核に注入し す Laboratory Press. 1986, Pinkert. Et al. Transgenic animal, technology .. Academic press. Inc. 1994) was applied to fertilized eggs. In other words, after fixing the fertilized egg in a culture solution with a glass bit for holding, a solution 21 of the δ gene fragment for introduction is injected into the male pronucleus using a small glass pipet.
2種類の遣伝子導入用断片を含む溶液は、 これらの導入用断片を丁 Ε锾衝液 (0.25mM EDTA 、 5m Tris(pH7.4))に怒 ®して、 それぞれ 1.0 gZm lになる ように調製したものを用いた。  The solution containing the two fragments for transfection is prepared by adding these transfection fragments to a buffer solution (0.25 mM EDTA, 5m Tris (pH 7.4)), and adding 1.0 gZml each. Was prepared.
( 2) 逭伝子注入をした受精卵の移植 (2) 移植 Transfer of fertilized eggs with gene injection
注入操作後、 C02 インキュベーター內にて、 M 2培地で 2〜 3時間培養後、 '受 精卵を発情している雌成熟豚の卵管へ移植した。 移植は、 全身麻酔下にて開腹後、 卵管内移植により行った。 卵管移植後分娩満期まで飼育し、 産仔を得た。 After the injection operation, in C0 2 incubator內, after 2-3 hours at M 2 medium, the 'fertilized egg was transplanted to the oviduct of a female adult pigs that estrus. Transplantation was performed by intratubal transplantation after laparotomy under general anesthesia. After tubal transplantation, the animals were bred to term until delivery, and pups were obtained.
なお、 遣伝子注入した受精卵は、 速やかに移植されるのが好ましい。  The fertilized eggs into which the gene has been injected are preferably transplanted immediately.
(実施例 5) (Example 5)
遣伝子導入ブタに於ける導入 ¾伝子の同定  Introduction in transgenic pigs 同 定 Identification of the gene
分娩後約 1週目に、 得られた仔プタの尾都の先端部分約 I cmを切断採取した。 尾部の細胞より高分子 DNAを抽出 ·精製した後、 サザンプロッティング法及び フィルターハイプリダイゼィション法によって、 導入した逸伝子断片がプタの染 色体に組み込まれているかを調べた。 即ち、 制限酵素 EcoR〖 と BamHI で完全に消 化させた 10 gの高分子 DN Aを 1 %ァガロースゲル鼋気泳動した後、 ナイロン フィルター （HybondN, Amersham)に DN Aを転写した。 フィルタ一は風乾後、 U V照射を行い DN Aを固定させて、 フィル夕一ハイプリダイゼーションに供した ( なお、 プローブとして、 pMAM/FTを制限酵素 Cla 【 と Nhe I で完全に消化 させた断片を FT用に用いた。 これらの桔果を下記表 1に示した。 計 1 9回の実驗で 5 5 0個の受精卵に ¾伝 子注入を行い、 同数を 1 9頭のレシビエントに移植した。 この結果 7頭か妊娠し， 2 7頭の仔ブ夕を分娩した。 これらのうち、 サザンプロッティング法及びフィル 夕一ハイブリダィゼイシヨン法によって、 FTに関して 1頭が 性であった。 な お、 遗伝子導入していないコン トロールブタ 1 5頭に対しても同様の検索を行つ たが、 いずれも陰性であった。 Approximately 1 week after parturition, about 1 cm of the tip of the obtained pupa was cut and collected. After extracting and purifying high-molecular-weight DNA from the cells in the tail, it was examined by Southern blotting and filter hybridization whether the introduced gene fragment was incorporated into the chromosome of the septum. That is, 10 g of high-molecular-weight DNA completely digested with restriction enzymes EcoR 〖and BamHI was subjected to 1% agarose gel electrophoresis, and then transferred to a nylon filter (HybondN, Amersham). Filter one is air dried, thereby fixing the DN A performs UV irradiation, Phil evening was subjected to a High Priestess die internalized (Note that fragment as a probe, to completely digest the pMAM / FT with restriction enzymes Cla [with Nhe I These results were shown in the following Table 1. In a total of 19 experiments, 550 fertilized eggs were transfected with a gene, and the same number was transferred to 19 recipients. As a result, seven of them became pregnant, We delivered 27 pups. Of these, one was sexually related to FT by the Southern plotting method and the Phil Yuichi Hybridization method. A similar search was performed on 15 control pigs into which no gene had been introduced, but all were negative.
【表 1】 【table 1】
Figure imgf000024_0001
Figure imgf000024_0001
(実施例 6) (Example 6)
導入遗伝子の発現の確認  Confirmation of gene expression
遣伝子導入か確認されている仔ブタの尾部の先端を約 1 cm切断し、 また血液を 採取し、 導入遺伝子発現の検出の試料とした。 採取した尾部より AGPC法（Aci d Guanidinium Thiocyanate-Phenol -Chloroform Method) を用いて、 全 RNAを 分離した。 次にこの全 RNAを使った RT - PCR法によって、 FTS伝子の発 現の解折を行った。  The tip of the tail of the piglet, which was confirmed to be transgenic, was cut about 1 cm in length, and blood was collected for use as a sample for detecting transgene expression. Total RNA was isolated from the collected tail using the AGPC method (Acid Guanidinium Thiocyanate-Phenol-Chloroform Method). Next, the expression of the FTS gene was analyzed by RT-PCR using this total RNA.
即ち、 全 RNAから逆転写酵素によって c DNAを合成し、 前述の FT遗伝子 抽出のために用いたブライマーを用いて PCR法を施行した。 その結果、 27頭 中 1頭が 1¾性と判定された。 すなわち、 導入した FT退伝子が mRNAに翻訳さ れ、 FTの酵素タンパク質を産生していることを確認した。 なお、 ¾伝子導入し ていないコン トロールブタ 1 5頭に対しても同様の検索を行ったが、 いずれも陰 性であつた。  That is, cDNA was synthesized from total RNA by reverse transcriptase, and PCR was performed using the primer used for FT gene extraction described above. As a result, one of 27 animals was determined to be 1¾. That is, it was confirmed that the introduced FT gene was translated into mRNA and produced an FT enzyme protein. A similar search was performed on 15 control pigs into which no gene had been introduced, but all were negative.
(実施例 7) (Example 7)
実施例 6で瞜性と判定された、 即ち FTの蛋白質を産生していると考えられる 仔プタから血液を採取し、 心後、 血球を分鶴し、 蛍光抗体を使った F I TC法 でブタ血球を染色した。 一方、 コントロールブタ （遣伝子導入していないプ夕） からも血液を採取し、 同様に染色した。 It was determined to be positive in Example 6, that is, it is considered to be producing FT protein Blood was collected from the pupa pupa, and after the heart, blood cells were separated and swine blood cells were stained by the FITC method using a fluorescent antibody. On the other hand, blood was also collected from control pigs (producers without transgenes) and stained similarly.
即ち、 一次抗体として抗 H型抗体を、 2次抗体として蛍光色素がラベルしてあ るマウス 7グロブリン （F(ab)2) を用いた。 この結果、 実施例 6の陽性の仔ブタ は陽性と判定された。 一方、 コン トロールブ夕のそれは全て陰性だった。 即ち、 導入された FTがブ夕钿胞の中で正しく機能していることが確かめられた。 That is, an anti-H-type antibody was used as a primary antibody, and mouse 7 globulin (F (ab) 2 ) labeled with a fluorescent dye was used as a secondary antibody. As a result, the positive piglets of Example 6 were determined to be positive. On the other hand, it was all negative in the control evening. That is, it was confirmed that the introduced FT was functioning correctly in the cell.
(実施例 8) (Example 8)
ブ夕の腎由来の細胞系 PK 1 5に pMAMZFTを感染させたコロニーを選択 し、 51C rを加えた培養液中にて、 一定時間 ¾養し、 その後健康な成人ヒ ト血液 (A, B. 0、 ABの 4つの血液型を含む） から採取した血漿を加えることによ り、 5'C rリ リースアツセィを行った。 A colony in which pMAMZFT was infected with the cell line PK15 derived from bushus kidney was selected and cultured for a certain time in a culture medium supplemented with 51 Cr, followed by healthy adult human blood (A, B. 0, Ri due to the addition of four including the blood type) plasma collected from the AB, 5 'was C r Li Risuatsusi.
この結果、 図 9に示すように、 14時間経過の後、 放出された C rを fflll定 した。 その結果、 コン トロールプタの血液はほぼ 1 00 ¾袖胞が破壊されたのが 観察されたが、 FTを導入したブタの細胞は 24時間経っても細胞破壊は 1 οκ 以下しか観測されなかった。  As a result, as shown in FIG. 9, after 14 hours had passed, the released Cr was determined as fflll. As a result, it was observed that the blood of the control ptera was destroyed by about 100 ¾ cysts, but the cells of the pigs transfected with FT showed less than 1 οκ even after 24 hours.
二のように、 プ夕細胞に FTを導入することによりいわゆる血液型が転換され, その結果ヒ 卜の 0型と同じ H型物質が生成されることになり、 ヒ トが持つプ夕に 対する自然抗体と抗原抗体反応を形成しないか、 あるいは抗原抗体反応を形成し ても低頻度であることが示された。 As shown in Fig. 2, by introducing FT into the cells, the so-called blood type is converted, and as a result, the same H-type substance as the human type 0 is produced. It was shown that the antibody did not form an antigen-antibody reaction with the natural antibody, or that the formation of the antigen-antibody reaction was infrequent.
(実施例 9 ) (Example 9)
相同 粗 田; 子》会 - ラ 1； ド ( T7FT yr)樣 ス 》伝芊断 L-GT,'FT) の調製 Preparation of transgenic L-GT, 'FT)
以下、 マウスの例を示すがブタにおいても基本的な考え型は同じである'  In the following, an example of a mouse is shown, but the basic idea type is the same in a pig as well.
( 1 ) フラグメント A(fr¾ment A)の樣築  (1) Construction of fragment A (fr¾ment A)
以下， フラグメント A(fragnientA)の構築を図 10に基づいて説明する.  The construction of fragment A (fragnientA) is described below with reference to Fig. 10.
既報 (Proc.Natl.Acad.Sci.USA. 86(1989), pp.8227-8231)のマウスの GT[EC2.4.1.151]の配列 に基づいてェキソン 2からェキソン 3までの部分を PC 法にて抽出するために. ェキソン 2の核酸配列に基づいてブラィマー A(primer A) [配列番号 3〗を、 又ェキソン 3の核酸配列 に基づいてブラィマー8 (pnmer B) [配列番号 4 ]を合成した。 これらのブラィマーと TaKaRa LAPC it Ver.2 (宝酒造；以下、 単に LA PCRと略す） とを用いて、 ェキソン 2からェ キソン 3までの部分のおよそ 1 O kbpを増幅した， 得られた断片を精製分離した後これを 铳型にしてブライマー A (primer A)とブライマー (primer Β') [配列番号 5 ]を用いて同社指 定のマニュアルに從つて LAPCRを施行した （以下ブラスミドへの組み込みに際しては各 製造社指定の方法により行うものとする） 。 ブライマー B'(prim B')には制限酵素 Sai lの 切断部位を含む， 得られた断片を精製分雜した後、これをブラスミ ド (pGEM-T; Proniega社） に組み込み、 これをブラスミ ド A(pi«mid A)と称した。 これを制限酵素 Sph lを含む種々の 制限酵索で切断して、 ェキソン 3の上流約 4.81cbpの部位に制限酵素 Sph lの切断部位が 1 Sf 所存在することが判明した。 そこでブラスミ ド Aを制限酵素 Spil lと制限酵素 Sai lとで切断 し、 約 Slcbpの断片を得た. これを精製分離した後、 T4 DNA polymerase (宝酒造)を用い て上記断片を平滑末端にした， これを断片 A(fr¾ineiitA)と称した。 86 (1989), pp.8227-8231, mouse exon 2 to exon 3 based on the sequence of mouse GT [EC2.4.1.151]. Synthesize Bramer A (primer A) [SEQ ID NO: 3〗 based on the nucleic acid sequence of exon 2 and Synthesize Bimer 8 (pnmer B) [SEQ ID NO: 4] based on the nucleic acid sequence of exon 2 did. Using these primers and TaKaRa LAPC it Ver.2 (Takara Shuzo; hereinafter simply abbreviated as LA PCR), amplify approximately 1 Okbp of exon 2 to exon 3 and purify the resulting fragment. After separation, it was converted to type 铳 and subjected to LAPCR using the primer A (primer A) and the primer (primer Β ') [SEQ ID NO: 5] according to the manual specified by the company. It shall be performed by the method specified by each manufacturer). The Buraima B '(prim B') comprising a cleavage site for the restriction enzyme Sai l, The resulting fragment was purified fraction雜, this Burasumi de; built into (pGEM-T Proni e g a company), which Was called Brasmid A (pi «mid A). This was cleaved with various restriction enzymes including the restriction enzyme Sphl, and it was found that there was 1 Sf of the restriction enzyme Sphl cleavage site at about 4.81 cbp upstream of exon 3. Then, the plasmid A was cleaved with restriction enzymes Spill and Sail to obtain a fragment of about Slcbp. After purification and separation, the fragment was blunt-ended using T4 DNA polymerase (Takara Shuzo). This was called fragment A (fr¾ineiitA).
( 2 ) フラグメント B(iragmenc B)の搆築  (2) Construction of fragment B (iragmenc B)
以下、 フラグメント B(irftgment B)の構築を図 11に基づいて説明する， Will be described below with reference to FIG. 11 the construction of fragment B (irftgm e nt B),
既報 (Proc.Nttl.Acad.Sci.じ SA, 86(1989), ρρ.8227-8231)のマウスの GT[EC2.4丄 151]の配列 に基づいてェキソン 4からェキソン 6までの部分を PCR法にて抽出するために、 ェキソン 4の核酸配列に基づいてブライマー C(primer C) [配列番号 6 ]を > 又ェキソン 6の核酸配列 に基づいてブライマー D(primer D) [配列番号 7 ]を合成した， これらのブライマーと LA-PCR とを用いて、 ェキソン 4からェキソン 6までの部分のおよそ 7 kbpを増幅した。 得られた 断片を精製分離した後. PCR from exon 4 to exon 6 based on the sequence of the mouse GT [EC2.4 の 151] of the previous report (Proc.Nttl.Acad.Sci. SA, 86 (1989), ρρ.8227-8231) to extract at law, Buraima C (primer C) based on the nucleic acid sequence of Ekison 4 Buraima D (prim er D) based on the nucleic acid sequence of [SEQ ID NO: 6]> the Ekison 6 [SEQ ID NO: 7] These primers and LA-PCR Was used to amplify approximately 7 kbp of the portion from exon 4 to exon 6. After purifying and separating the obtained fragments.
これをブラスミ ド J>GEM-T (Promega社)に組み込み、 これをブラスミ ド B(piumid B)と称し た， これを 限 ® Nco Iを含む種々の制限酵素で切断して. 上記断片に制限酵素 Nco lの 切断部位が一箇所存在することが判明した。 又制限酵素 Sec IIを含む種々の制限酵素で切 断して， も上記断片に制隨素 Sac Πの切断部位が一面所も^ Sせす、 ブラスミ B This was incorporated into Brasmid J> GEM-T (Promega), and this was called Brasmid B (piumid B), which was cut with various restriction enzymes including Nco I. Restriction to the above fragment It was found that there was one cleavage site for the enzyme Ncol. In addition, after cutting with various restriction enzymes including the restriction enzyme Sec II, the cleavage site of the constitutive element Sac せ is also added to the above fragment by ^ S.
(plasmid B)に存在する制限酵素 Sac IIの切断部位はブラスミ ド pGEM-T( Promega社)の 5'側の クローニング部位のみであることが判明した. そこでブラスミ ド Bを制限薛素 Sac IIと Nco Iとで切断し， 約 3. bpの靳片を得た。 これを搰製分離した後. T4 DNA polymerase (宝酒 造)を用いて上記断片を平滑末端にした。 この断片に対し. ヌクレオチドの丁で½まる Sac Πリンカ一 (New Eflland Bioiab nc.)付加した。 これを靳片 B (fragmem B) と称した，(plasmid B) was found to be restricted to the cloning site on the 5 'side of the plasmid pGEM-T (Promega). The fragment was cut with NcoI to obtain a fragment of about 3. bp. After this was isolated from the manufacturer, the above fragment was blunt-ended using T4 DNA polymerase (Takara Shuzo). To this fragment was added Sac linker (New Eflland Bioiab nc.), Which was filled with nucleotides. This was called fragment B (fragmem B).
( 2 ) プラスミド C(plasmid C)の構築 (2) Construction of plasmid C
以下、 ブラスミ ド C(plasmid C)の構築を図 1 2に基づいて鋭明する，  Hereinafter, the construction of brassmid C (plasmid C) will be sharpened based on FIG.
et$g(Proc.Naa. Acad. Sci. USA, 87(1990), pp.6674-6678)のヒ卜の FT[EC2.4.1.9(S】の DNA配 列に基づいてェキソン 1力、らェキソン 2までの部分の cDNAを RT-PCR法にて抽出するため に、 ブライマー E(primer E) [配列番号 8 ]とブライマー F(primer F) [配列番号 9 ]を合成した， この cDNAの合成の方法については上記実施例 1に記載した通りである。 これらブライマ 一を用いて施行された RT-PCR法により得られた断片 （これを断片 Cと称する） には FTの 発現のためのスタートコドンとストップコドンが含まれている。 又スタートコドンの上流 には制限酵素 Sal Iの切断部位が存在している.  USA, 87 (1990), pp. 6674-6678), and the FT of the human [exon 1 force based on the DNA sequence of EC2.4.1.9 (S), et $ g (Proc. Naa. Acad. Sci. Primer E (SEQ ID NO: 8) and Primer F (primer F) [SEQ ID NO: 9] were synthesized to extract the cDNAs from exon 2 through RT-PCR. The method of synthesis is as described in Example 1 above.A fragment obtained by the RT-PCR method (referred to as fragment C) carried out using these primers was used for the expression of FT for expression of FT. It contains a start codon and a stop codon, and a cleavage site for the restriction enzyme Sal I exists upstream of the start codon.
次に， この断片 (fra ment ed . 発現ベクター pCR3(Invitrogen社)に組み込み、 これをブラ スミ ド C(plasmid C)と称した t Then, this fragment (fra ment ed. Incorporated into an expression vector pCR3 (Invitrogen, Inc.), was designated At the bra corner de C (plasmid C) t
( 3 ) ブラスミ ド D(piasmid D)の構築  (3) Construction of brasmid D
以下、 プラスミド D(piasmi(J D)の構築を図 12に基づいて説明する，  Hereinafter, the construction of plasmid D (piasmi (JD) will be described with reference to FIG.
上記発現べクタ一: pCR3の 1113番のヌクレオチドの部位に、 即ち pCR3の poiy Aの部位の すぐ下流に制限酵素 ΑΠ〗11が唯一存在し. 又この制限酵素は FTを含む断片 C(frtgment C)を 切断しないこと力 かめられている. そこでブラスミ ド C(plasmid C)を制限酵素 ΑίΠΠで 切断した， これを前 |gT4DNApoiyine e (宝酒造)を用いて上記断片を平滑末端にした. 次にこの断片に前記 Sac IIリンカ一 (Mew Enlwid Biolabs, c.)を付加した。 これを制限酵素 SacIIで切断した後、 これを前記 WDNAiigase (宝酒造)を用いて self-ligation 処理した。 この結果制限酵素 Sac IIのすぐ下流に新たに制限酵素 ΑΠ ΠΙの切断部位が出現する， この 断片をブラスミ ド D(phsmidD)と称した， The above expression vector: The only restriction enzyme ΑΠ〗 11 is present at nucleotide position 1113 of pCR3, that is, immediately downstream of the poiy A site of pCR3. This restriction enzyme is a fragment C containing FT (frtgment C ) Is cut off. Therefore, plasmid C is restricted with restriction enzyme C. Cut, this before |. GT4DNApoiyin with ee (Takara Shuzo) was blunted with the fragments then the this fragment Sac II linker one (Mew Enlwid Biolabs, c.) Was added. This was cut with the restriction enzyme SacII, and then subjected to self-ligation treatment using the aforementioned WDNAiigase (Takara Shuzo). As a result, a new cleavage site for the restriction enzyme ΑΠ 出現 appears immediately downstream of the restriction enzyme Sac II. This fragment was called plasmid D (phsmidD).
(4 ) ブラスミ ド E(piasmid E)の構築  (4) Construction of brassmid E (piasmid E)
以下. ブラスミ ド E(piasmidE)の擐築を図 13に基づいて説明する，  The construction of Brasmid E (piasmidE) is described below with reference to FIG.
上記ブラスミ ド ！！^り 制限酵素 ^で処理し. これを脱リン化酵素 (CIAP:宝滔 造)で脱リン化処理した後， これに. 制限酵素 5 11で処理した断片8 ^««8)を前記丁4 DNAligase (宝酒造)を用いて ligation処理した。 これをブラスミ ド E(piasmid E)と称する。  Brasmid above! ! This is treated with a restriction enzyme ^. This is dephosphorylated with a dephosphorylase (CIAP: Takatozo), and then the fragment 8 ^ «« 8) treated with restriction enzyme 5 11 is 4 Ligation was performed using DNAligase (Takara Shuzo). This is called Brasmid E.
(5) ブラスミ ド FpiasiwtJ F)の構築  (5) Construction of Brasmid FpiasiwtJ F)
以下. ブラスミ ド F(piasmiciF)の樣築を図 Wに基づいて説明する。  The construction of Brasmid F (piasmiciF) is described below with reference to Fig. W.
前記ブラスミド E(plasmidE)を制限酵素 Sailで切断し平滑化処理した後、 前記丁 4 DNA ligese (宝酒造)を用いて断片 A(ir meiitA)と ligation処¾した， これをブラスミ ド F(piasmid The plasmid E was cut with the restriction enzyme Sail, blunt-treated, and ligated with the fragment A (ir meiitA) using the DNA ligese (Takara Shuzo).
F)と称した. F).
(6) コンストラクトの棣築  (6) Construction of Di
以下. コンストラクトの構築を図 Wに基づいて説明する。  The construction of the construct is described below with reference to FIG.
上記ブラスミド F(plasmid F)を制限酵素 Sai I及 Ail IIIで切断した。 これにより約 10 kbpの 長さの断片を得た。 これを前記のように分雜糟製し， 港度を 5.0μ8/μ1に調整し、 ； g伝子導 入用の适伝子断片として、 レ στ/πと称した， The above plasmid F was cut with restriction enzymes Sai I and Ail III. This resulted in a fragment approximately 10 kbp in length. This was manufactured as described above, and the portability was adjusted to 5.0 μ 8 / μ1; as a 适 gene fragment for introducing g gene, it was referred to as στ / π.
(7) コンストラク卜の特遛  (7) Features of the construction
以下 このコンストラクトの特徴を述べる，  The features of this construct are described below.
それはまず第一に， このコンストラクトは. GTの ¾伝子の翻訳開始コードを含む配列部 分を欠失する形で変異配列が挿入されていることである， 第 2に， この押入された配列が、 FTの遼伝子をコードする翻訳部分 （翻訳關始コードおよび翻訳停止コードを含む） をす ベて含んでいることである， 第 3に、 前記押入された配列が. FTの翻訳部分の 3' 側に ターミネータ一を含む点である。 第 4に. 前記神入された配列が、 GTの ¾伝子の翊訳開 δ コードの 5 ' 側の非 ®訳部分と結合していることである， 第 5に. 前記抻入された配列 の部分の長さが. 内在する GTの适伝子の欠失する部分の長さとほほ同じであること. 言 い替えればコンストラク卜の配列の長さが、 內在する GTの逸伝子の中の相同組み換えを 起こす δβ分の長さとほぼ同じであることである。 First of all, this construct has a mutant sequence inserted in a form that deletes the sequence portion containing the translation initiation code of the .GT gene. Secondly, this inserted sequence Contains all the translation parts (including the translation start code and translation stop code) that encode the FT Liao gene. Third, the indented sequence is the .FT translation part. 3 'is to include a terminator. Fourth. The god-introduced array is a translator of GT's 子 伝 子 It is linked to the untranslated part on the 5 'side of the δ code. Fifth. The length of the part of the inserted sequence is reduced. In other words, the length of the sequence of the construct is almost the same as the length of δβ that causes homologous recombination in the existing gene of GT.
これら特徴の結果、 比較的， 相同組み換えが起こりやすく . 力つ相同組み換え力《起きた場 合、 GTの igi云子は詡訳開始コードを欠くために GTが発現せす， かつ FTの逍伝子が IS訳さ れ発現することになる。  As a result of these characteristics, homologous recombination is relatively likely to occur. If the homologous recombination force occurs, the GT will be expressed due to the lack of the translation start code in the GT igi cloud, and the FT will follow The child is translated and expressed.
(実細 1 0 ) (Actual 10)
Figure imgf000029_0001
Figure imgf000029_0001
マウスの培 細胞し 9 2 9を直径 3 cmの培養皿内でおよそ 2 mlの血滑培地 （FCS加  In a 3 cm-diameter culture dish, add about 2 ml of blood culture medium (FCS
RPMI)の中でおよそ 1.0x10s個の钿胞が認められるようになるまで培養し、 この日を第 1日 目とすると. 第 2日目には無血滑培地に替え、 約 3時間の後に次に述べる溶液を上記培養 皿に添加した。 即ち. A液として、 7μ1の DMEM溶液に前記遺伝子断片レ GT/FTを 2μ1, 及び 前記 pMAM w(l.(^g^i)を ΐμΐ加えた混合液を用意した。 Β液として. 5μ1の DMEM溶液に， RPMI) until about 1.0x10 s cells are observed, and this day is the first day. On the second day, change to a serum-free medium and after about 3 hours The following solutions were added to the culture dishes. That is, a mixed solution was prepared by adding 2 μl of the gene fragment GT / FT and 7 μl of the pMAM w (l. (^ G ^ i) to the 7 μl DMEM solution to the 7 μl DMEM solution. In the DMEM solution,
¾伝子導入用合成脂質であるリボフェクチン （Gibco社） 5μ1を加えた混合溶液を用意した， この混合溶液 Αを混合溶液 Βに加えた. 室 ¾|にて約 1 5分培赛し、 DMEM溶液を 30μ!加え， 静かに混合した上で. 上記マウスの培養細胞 L- 9 2 9が培養されている培養皿へ添加した。 第 3日目の朝には 1095 FBS加 PRMI溶液を l.Sml加え， 次の朝まで培養した， 第 4日目の朝 には培蹇液をすベて 2mlの 10« FBS加 P MI溶液に谊き換え， その後更に 2日培養した. 第 6日目にはバサージュして逭径 1 0 cmの培養皿にすべての細胞を移し、 皿に 1日培養し た。 第 7曰目には、 G418を最終膿度 300 g/miになるように調整した 10« FBS加 P MI溶液 混合 A mixed solution containing 5 µl of ribofectin (Gibco), a synthetic lipid for introducing a gene, was prepared. This mixed solution Α was added to mixed solution .. The solution was added in an amount of 30 μ !, and the mixture was gently mixed. The solution was added to a culture dish in which the cultured mouse cells L-929 were cultured. On the morning of the third day, add l.Sml of 1095 FBS plus PRMI solution and incubate until the next morning. Then, the cells were further cultured for 2 days. On the 6th day, all cells were transferred to a culture dish having a diameter of 10 cm by bassage and cultured on the dishes for 1 day. The seventh statement is that G418 was adjusted to a final pus level of 300 g / mi.
1 O mlで培養液を Sき換えた加えた， この後さらに 7日培養し. 前記 G418加 1095 FBS加 PRMI溶液 1 0 mlで培溶液を Sき換えた， The medium was replaced with S at 1 O ml, and the culture was further cultured for 7 days. The medium was replaced with 10 ml of the PRMI solution with 1095 FBS and 10 ml of G418.
(宾施例 1 1 )  (宾 Example 1 1)
；眚 ¾ λマ ス細½1に ¾Η十 A A ;W ネの m  眚 λ λ mass detail 1 ½ 10 A A; W m
こうした結果， 培蹇皿の中に 1 8 7個のコロニーを得た。 得られたコロニーをバサージュして更に增? iさせてから細胞を抽出した， 前記方法にて DNAを抽出できた細胞群が 1 5 2個たつた. これらの D Aを制限酵素 Sph lと BgUIで切 断した. 前記方法により Southern blottin を行った， probeとして FTの cD Aを用いた As a result, 187 colonies were obtained in the culture dish. The obtained colonies were subjected to a vasage and further extracted, and the cells were extracted. There were 152 cells in which DNA could be extracted by the above-mentioned method. These DAs were separated with restriction enzymes Sphl and BgUI. Southern blottin was performed by the method described above, using FT cDNA as a probe.
Southera blowing結果， 1 4 2個の細胞群が |¾性だった （図 16A) c これらのうち、 図 15に 示す様に本来存在しない外来 ifii云子 FTの配列の中から igんたブライマー G [配列番号 1 0】 及 H【配列番号 1 1 ]を用い前記の様に LA-PCRを施行した. 即ち、 同所性組み換え As a result of Southera blowing, 14 2 cell groups were ¾-positive (Fig. 16A). C Among these, as shown in Fig. 15, the primer ig was selected from the non-existent foreign ifii-union FT sequence. LA-PCR was performed using [SEQ ID NO: 10] and H [SEQ ID NO: 11] as described above.
(homologous recombiriftuoii ) が起こっているときにだけブライマー Aとブライマー Gによ る PC において約 1 O kbpの長さのパンド. 及ブライマー Dとブライマー Hによる PC にお いて約 7 kbpの長さのバンドを得る。 実際上記 1 4 2個の細胞群のうち 3個の細胞群にお いてこれらの LA-PCRにおいて陽性だった （1U6Bおよび 16C) . 同所性組み換えが起こつ ている時以外ではこれらのバンドが検出されないことは明らかである （図 16Bおよび 16C (1 kbp length band in PC by primers A and G only when (homologous recombiriftuoii) is occurring. And band of about 7 kbp length in PC by primers D and H. Get. In fact, three of the 142 cell groups were positive in these LA-PCRs (1U6B and 16C). These bands were not present except when orthotopic recombination had occurred. Clearly not detected (Figures 16B and 16C
(実施例 1 ) (Example 1)
導 λ ^伝子の発現の確認  Confirmation of expression of the derived λ ^ gene
前記の;き ί云子導入絪胞を更に培養し. 全 NAを抽出した。 これを基に前記ブライマー Ε 及 Fを用いて RT-PCRを施行した， この結果、 前記 3細胞群の内 3細胞群に於いて RT-PCR 上！ ¾性であった （図 16D) 。 また、 ¾光檁 ISがラベルされているレクチン （UEA-1; EY bolatones) を用いて、 上記外来 ¾伝子細胞群および非導入細胞群を染色し， フローサイ トメトリーにて前記 H抗原の発現を確認した （図 16Eおよび F) 。 なお、 図 16Eの L929 / Neo / UEA-Iとは. ネオマイシン耐性 ifi伝子を導入したマウス LS29細胞群を前記レクチン UEA-Iを用いて H抗原の発現の程度を測定したことを示す。 同様に図 1I5Fの L929 I FT ί UEA-Iとは. 前記外来道伝子 L-GT/FTを導入したマウス L929細胞群を前記レクチン UEA-I を用いて H抗原の発現の程度を測定したことを示す。  The above-mentioned transduced cells were further cultured. The total NA was extracted. Based on this, RT-PCR was performed using the above primers and F. As a result, three out of the three cell groups were subjected to RT-PCR! It was positive (Fig. 16D). In addition, the exogenous gene group and the non-transfected cell group were stained with a lectin (UEA-1; EY bolatones) labeled with ¾IS, and the expression of the H antigen was determined by flow cytometry. Confirmed (Figures 16E and F). In addition, L929 / Neo / UEA-I in FIG. 16E indicates that the expression level of H antigen was measured using the lectin UEA-I in a mouse LS29 cell group into which the neomycin-resistant ifi gene was introduced. Similarly, FIG. 1 shows L929 I FT ί UEA-I of I5F. The degree of H antigen expression was measured using the lectin UEA-I in the mouse L929 cell group into which the exogenous pathogen L-GT / FT was introduced. Indicates that
これらの結果は明らかに外来導入通伝子 L-GTZFTがマゥスの体細胞の中に同所性に組み 換えられていること， かつ導入された FTが発現していることを意味している. このこと は、 もしこの導入外来用 ¾伝子 L-GT/FTが生 細昀系列に ¾入されていれば、 しかるべ き継代を経て， 外来 iSi云子 L-OT/FHこ閟してホモの伏悲になりうること， すなわち G抗 原の代わりに H抗原を発現している個体の出現が期待できることを示している。 (実施例 1 3 ) These results clearly indicate that the exogenous transgene L-GTZFT is orthotopically recombined in mouse somatic cells and that the transduced FT is expressed. This means that if the introduced exogenous gene L-GT / FT has been introduced into the production line, it must be passed through a certain passage before exogenous iSi-union L-OT / FH cogeneration. This indicates that homozygosity can result in homozygosity, that is, the appearance of individuals expressing the H antigen instead of the G antigen can be expected. (Example 13)
港入照 子断 （PGT / FT) のマウス細 gftへの堪 λ の Igl??お上び のお ¾の 導入用道伝子断片 （pGT / FT) のマウス細胞への導入については上記実施例 1 0に準じ て行った. その同定についても上記実施例 1 1に準じて行った，  Transfection of the transgenic fragment (pGT / FT) into mouse cells using the transfection gene (pGT / FT) for Igl ?? The identification was performed according to Example 11 described above.
こうした转果， 培養皿の中に 3 6 1個のコロニーを得た， As a result, we obtained 361 colonies in the culture dish.
得られたコロニーをバサージュして更に增¾させてから細胞を抽出した， 前記方法にて DNAを抽出できた細胞群が 2 8 5個だった。 これらの DNAを锞型にしてブライマー （P, C) および （pZD) を用いて PCRを施行した， その結果. 1 3 7個の細胞群が！ ¾性だった (図 17A) · これらのうち， 本来存在しない外来适伝子 FTの配列の中から jgんだブライマ 一および内在する GTにかかわる部分から遴んだブライマーを用いて PC を施行した. 上記 1 3 7個の細胞群のうち 1個の細胞群において腸性のバンドを検出した （図 17Bおよ び 17C) , The obtained colonies were subjected to bassage to further elongation, and then the cells were extracted. The number of cells from which DNA could be extracted by the above method was 285. Using these DNAs as type III, PCR was performed using primers (P, C) and (pZD). As a result, 13 7 cell groups! (Fig. 17A) · Among these, PC was performed by using a jig from the sequence of the non-existing exogenous gene FT and a primer from the part related to the intrinsic GT. Intestinal bands were detected in one of the 1337 cell groups (Figures 17B and 17C),
次にこの細胞群から全 RNAを抽出し， R丁- PC を施行した。 その結果、 陽性のパンドを得 た （図 17D) 。 次に前記のレクチンを用いて前記の細胞の H抗原の発現についてフローサ ィ トメトリーを用いて検定し、 ¾性の結果を得た （図 17Eおよび図 17F) . Next, total RNA was extracted from this cell group and subjected to R-PC. As a result, a positive band was obtained (Fig. 17D). Next, the expression of H antigen in the cells was assayed using flow cytometry using the lectin, and positive results were obtained (FIGS.17E and 17F).
一 3 ひ一 One 3 Hiichi
なお、 この発明は、 その本贫的特性から逸脱することなく数多くの形式のもの として具体化することができるから、 これらの実施 様はもっぱら説明上のもの で制約的なものではない。 また、 この ¾明の IS囲は、 請求の IB囲以外の記裁によ るものでなく、 請求の範囲によって限定するものであるから、 請求の β囲の要件 内のあらゆる変更、 またはその要件に対する均等物は請求の範囲に包含されるも のである。 It should be noted that the present invention can be embodied in many forms without departing from the essential characteristics thereof, and thus these embodiments are merely illustrative and not restrictive. In addition, since the IS box in this description is not determined by a claim other than the IB box in the claim and is limited by the scope of the claim, any change in the requirements in the β box of the claim or its requirement The equivalents to are intended to be covered by the appended claims.
配列表 1 ) 一般情報 Sequence Listing 1) General information
( i ) 出願人 小池 千裕  (i) Applicant Chihiro Koike
(ii ) 発明の名称： 外来遺伝子の導入により高等霊長類の抗原型を発現した非 ¾ 霊長哺乳類の形質転換動物及びその作出方法  (ii) Title of the Invention: Transgenic non-¾ primate mammal expressing higher primate serotype by introduction of a foreign gene and method for producing the same
(ώ)配列の数： 1 1 (ώ) Number of arrays: 1 1
Civ) 連格先住所： Civ) Reputation address:
CA) 宛て名 ：小池 千裕  CA) Address: Chihiro Koike
(B) 通り ：八竜町 1一 3 0  (B) Street: Hachiryu-cho 1 1 3 0
(C) 市：名古星巿  (C) City: Nagoboshi II
(D) 州：愛知県  (D) State: Aichi Prefecture
(E) 国： 日本  (E) Country: Japan
(F) 郵便番号： 462  (F) ZIP code: 462
( V ) コ ンピュータ読み取り可能形式 (V) Computer readable format
(A) 媒体方式：  (A) Medium system:
(B) コンビュ一タ  (B) Computer
(C)動作システム  (C) Operation system
(D) ソフ トウェア  (D) Software
(vi) 現行適用データ  (vi) Current applicable data
(A) 適用番号： - (A) Application number:-
(B) ファイル B ： (B) File B:
( C ) 分類：  (C) Classification:
(vii) 事前適用データ  (vii) Pre-application data
(A) 適用番号：  (A) Application number:
( B ) ファイル日 ：  (B) File date:
(C) 分類：  (C) Classification:
(νϊ) 了 トニー/エージェン ト情報  (νϊ) End Tony / Agent information
(Α) 氏名 ： (B) 登錄番号： (Α) Name: (B) Registration number:
(C) 参照/事件番号：  (C) Reference / case number:
(ix) 通信手段情報 (ix) Communication method information
(A) ¾話： 0 5 2 9 1 2— 7 0 6 2 (A) ¾ story: 0 5 2 9 1 2— 7 0 6 2
(B) ファクシミ リ ： 0 5 2/9 1 2— 7 0 6 2(B) Facsimile: 0 5 2/9 1 2—7 0 6 2
(C) テレッ クス ： (C) Telex:
(2) 配列番号 1 (SEQ 〖D N0:1)に閱する情報 (2) Information shown in SEQ ID NO: 1 (SEQ 〖D N0: 1)
( i ) 配列の特徴 ··  (i) Sequence features
(A) 長さ ： 1 1 74  (A) Length: 1 1 74
(B) 型 ：核酸  (B) Type: nucleic acid
(C) 鎖の数：二本鎖  (C) Number of chains: double-stranded
CD) トポロジー ：直鎖状  CD) Topology: Linear
(ii) 配列の種類 ·· c DNA t o mRNA  (ii) Sequence type ... c DNA t o mRNA
(A) 特徴 ：な（ し 2) フコシル トラ ンスフェラーゼ  (A) Features: N (2) Fucosyl transferase
Ciii) ハイボセティ カル ： No  Ciii) Hibosetical: No
(iv) アンチセンス ： No  (iv) Antisense: No
Cx) 刊行物情報：  Cx) Publication information:
(A) 著者： Larsen.R.D.  (A) Author: Larsen.R.D.
Ernest,し.  Ernest, then.
Nair.R.P.  Nair.R.P.
し owe. J. B.  Owe. J. B.
( B ) 理名 ： Molecular cloning, sequence, and expression of a  (B) Real name: Molecular cloning, sequence, and expression of a
Human GDP -し - iucose: - D-galactoside 2-alfa-L- fucosyltransferase cDNA that can form the H blood group antigen.  Human GDP -shi-iucose:-D-galactoside 2-alfa-L-fucosyltransferase cDNA that can form the H blood group antigen.
(C) 雑 名： Proceedings of the National Academy  (C) Miscellaneous name: Proceedings of the National Academy
of Science. USA  of Science. USA
(D) 巻数 ： 87  (D) Number of turns: 87
(F) 頁数 ： 6674-6678  (F) Number of pages: 6674-6678
(G) 日付： SEP-1990  (G) Date: SEP-1990
(xi) 配列の記載： SEQ I D NO : l :  (xi) Sequence description: SEQ ID NO: l:
CAAQCAGCTC GGCC 14CAAQCAGCTC GGCC 14
A G TCG CTC CGG AGC CAT CGT CAG CTC TGC CTG GCC TTC CTG 56 Met Trp Leu Arg Ser His Arg Gin Leu Cys Leu Ala Phe Leu A G TCG CTC CGG AGC CAT CGT CAG CTC TGC CTG GCC TTC CTG 56 Met Trp Leu Arg Ser His Arg Gin Leu Cys Leu Ala Phe Leu
1 5 10 - 3 4 - 1 5 10 -3 4-
CTA GTC TGT GTC CTC TCT GTA ATC TTC TTC CTC CAT ATC CAT 98 Leu Val Cys Val Leu Ser Val lie Phe Phe Leu Hie lie His CTA GTC TGT GTC CTC TCT GTA ATC TTC TTC CTC CAT ATC CAT 98 Leu Val Cys Val Leu Ser Val lie Phe Phe Leu Hie lie His
15 20 25 15 20 25
JA GAC AGC TTT CCA CAT GGC CTA GGC CTG TCG ATC CTG TGT 140 Gin Asp Ser Phe Pro His Gly Leu Gly Leu Ser He Leu Cye  JA GAC AGC TTT CCA CAT GGC CTA GGC CTG TCG ATC CTG TGT 140 Gin Asp Ser Phe Pro His Gly Leu Gly Leu Ser He Leu Cye
30 35 40  30 35 40
CCA GAC CGC CGC CTG GTQ ACA CCC CCA GTQ GCC ATC TTC TGC 182 Pro Asp Arg Arg Leu Val Thr Pro Pro Val Ala He Phe Cys  CCA GAC CGC CGC CTG GTQ ACA CCC CCA GTQ GCC ATC TTC TGC 182 Pro Asp Arg Arg Leu Val Thr Pro Pro Val Ala He Phe Cys
45 SO 55  45 SO 55
CTG CCG GGT ACT GCG ATG GGC CCC AAC GCC TCC TCT TCC TGT 224 Leu Pro Gly Thr Ala Met Gly Pro Asn Ala Ser Ser Ser Cys  CTG CCG GGT ACT GCG ATG GGC CCC AAC GCC TCC TCT TCC TGT 224 Leu Pro Gly Thr Ala Met Gly Pro Asn Ala Ser Ser Ser Cys
SO 65 70  SO 65 70
CCC CAG CAC CCT GCT TCC CTC TCC GGC ACC TOO ACT GTC TAC 2S6 Pro Gin His Pro Ala Ser Leu Ser Qly Thr Trp Thr Val Tyr  CCC CAG CAC CCT GCT TCC CTC TCC GGC ACC TOO ACT GTC TAC 2S6 Pro Gin His Pro Ala Ser Leu Ser Qly Thr Trp Thr Val Tyr
75 80  75 80
CCC AAT GGC CGG TTT GQT AAT CAG ATG GGA CAG TAT GCC ACG 308 Pro Aan Gly Arg Phe Gly Asn Gin Met Gly Gin Tyr Ala Thr  CCC AAT GGC CGG TTT GQT AAT CAG ATG GGA CAG TAT GCC ACG 308 Pro Aan Gly Arg Phe Gly Asn Gin Met Gly Gin Tyr Ala Thr
85 90 95  85 90 95
CTG CTG GCT CTG GCC CAG CTC AAC GGC CGC CGG QCC TTT ATC 350 Leu Leu Ala Leu Ala Gin Leu Aan Gly Arg Axg Ala Phe lie  CTG CTG GCT CTG GCC CAG CTC AAC GGC CGC CGG QCC TTT ATC 350 Leu Leu Ala Leu Ala Gin Leu Aan Gly Arg Axg Ala Phe lie
100 105 110  100 105 110
CTG CCT GCC ATG CAT GCC GCC CTG GCC CCG GTA TTC CGC ATC 392 Leu Pro Ala Met Hie Ala Ala Leu Ala Pro Val Phe Arg lie  CTG CCT GCC ATG CAT GCC GCC CTG GCC CCG GTA TTC CGC ATC 392 Leu Pro Ala Met Hie Ala Ala Leu Ala Pro Val Phe Arg lie
115 120 125  115 120 125
ACC CTG CCC GTG CTG GCC CCA GAA GTQ GAC AGC CQC ACG CCG 434 Thr Qu Pro Val Leu Ala Pro Glu Val Asp Ser Arg Thr Pro  ACC CTG CCC GTG CTG GCC CCA GAA GTQ GAC AGC CQC ACG CCG 434 Thr Qu Pro Val Leu Ala Pro Glu Val Asp Ser Arg Thr Pro
130 135 140  130 135 140
TGG CGG GAG CTG CAG CTT CAC GAC TGG ATG TCG GAG GAG TAC 476 Trp Arg Glu Leu Gin Leu His Λ5ρ Trp Met Ser Glu Glu Tyr  TGG CGG GAG CTG CAG CTT CAC GAC TGG ATG TCG GAG GAG TAC 476 Trp Arg Glu Leu Gin Leu His Λ5ρ Trp Met Ser Glu Glu Tyr
145 150  145 150
GCG GAC TTG AGA GAT CCT TTC CTG AAG CTC TCT GGC TTC CCC 51B Ala Asp Leu Arg Asp Pro Phe Leu Lys Leu Ser Gly Phe Pro  GCG GAC TTG AGA GAT CCT TTC CTG AAG CTC TCT GGC TTC CCC 51B Ala Asp Leu Arg Asp Pro Phe Leu Lys Leu Ser Gly Phe Pro
155 160 165 155 160 165
TGC TCT TGG ACT TTC TTC CAC CAT CTC CQG GAA CAG ATC CGC 560 Cys Ser Trp Thr Phe Phe His Hie Leu Arg Glu Gin lie Arg  TGC TCT TGG ACT TTC TTC CAC CAT CTC CQG GAA CAG ATC CGC 560 Cys Ser Trp Thr Phe Phe His Hie Leu Arg Glu Ginlie Arg
170 175 180  170 175 180
A6A GAG TTC ACC CTG CAC GAC CAC CTT CGG GAA GAG GCG CAG 602 Arg Glu Phe Thr Leu His Asp Hie Leu Arg Glu Glu Ala Qln  A6A GAG TTC ACC CTG CAC GAC CAC CTT CGG GAA GAG GCG CAG 602 Arg Glu Phe Thr Leu His Asp Hie Leu Arg Glu Glu Ala Qln
185 190 195  185 190 195
ΑΞΤ GTG CTG GQT CAG CTC CGC CTG GGC CGC ACA GGO SAC CGC 644 Ser Val Leu Gly Gin Leu Arg Leu Gly Arg Thr Gly Asp Arg  ΑΞΤ GTG CTG GQT CAG CTC CGC CTG GGC CGC ACA GGO SAC CGC 644 Ser Val Leu Gly Gin Leu Arg Leu Gly Arg Thr Gly Asp Arg
200 205 210 CCO CGC ACC TTT GTC GGC GTC CAC GTG CGC CGT GGG GAC TAT 6BG Pro Arg Thr Phe Val Gly Val Hie Val Arg Arg Gly Asp Tyr 200 205 210 CCO CGC ACC TTT GTC GGC GTC CAC GTG CGC CGT GGG GAC TAT 6BG Pro Arg Thr Phe Val Gly Val Hie Val Arg Arg Gly Asp Tyr
215 220  215 220
CTG CAG GTT ATG CCT CAG CGC TGG AAG GGT GTG GTG GGC GAC 72 B Leu Gin Val Met Pro Gin Arg Trp Lye Gly Val Val Gly Asp  CTG CAG GTT ATG CCT CAG CGC TGG AAG GGT GTG GTG GGC GAC 72 B Leu Gin Val Met Pro Gin Arg Trp Lye Gly Val Val Gly Asp
225 230 235 225 230 235
AGC GCC TAC CTC CGG CAG GCC ATG GAC TGG TTC CGG GCA. CGO 770 Ser Ala Tyr Leu Arg Gin Ala Met Asp Trp Fhe Arg Ala Arg  AGC GCC TAC CTC CGG CAG GCC ATG GAC TGG TTC CGG GCA.CGO 770 Ser Ala Tyr Leu Arg Gin Ala Met Asp Trp Fhe Arg Ala Arg
240 245 250  240 245 250
CAC GAA GCC CCC GTT TTC GTG GTC ACC AGC AAC GGC ATG SAG 812 His Glu Ala Pro Val Phe Val Val Thr Ser Asn Gly Met Glu  CAC GAA GCC CCC GTT TTC GTG GTC ACC AGC AAC GGC ATG SAG 812 His Glu Ala Pro Val Phe Val Val Thr Ser Asn Gly Met Glu
255 260 265  255 260 265
TGG TGT ΑΛΆ. GAA AAC ATC GAC ACC TCC CAG GGC GAT GTG ACG 854 Trp Cys Lys Glu Asn lie Asp Thr Ser Gin Gly Asp Val Thr  TGG TGT ΑΛΆ. GAA AAC ATC GAC ACC TCC CAG GGC GAT GTG ACG 854 Trp Cys Lys Glu Asn lie Asp Thr Ser Gin Gly Asp Val Thr
270 275 280  270 275 280
TTT GCT GGC GAT GGA CAG GAG GCT ACA CCG TGG AAA GAC ΊΤΓ 896 Phe Ala Gly Asp Gly Gin Glu Ala Thr Pro Trp Lys Asp Phe  TTT GCT GGC GAT GGA CAG GAG GCT ACA CCG TGG AAA GAC ΊΤΓ 896 Phe Ala Gly Asp Gly Gin Glu Ala Thr Pro Trp Lys Asp Phe
285 290  285 290
GCC CTG CTC ACA CAG TGC AAC CAC ACC ΑΊ ATG ACC ATT GGC 93 B Ala Leu Leu Thr Gin Cys Asn His Thx lie Met Thr lie Gly  GCC CTG CTC ACA CAG TGC AAC CAC ACC ΑΊ ATG ACC ATT GGC 93 B Ala Leu Leu Thr Gin Cys Asn His Thx lie Met Thr lie Gly
295 300 305 295 300 305
ACC TTC GGC TTC TGG GCT GCC TAC CTG GCT GGC GGA GAC ACT 980 Thr Phe Gly Phe Trp Ala Ala Tyr Leu Ala Gly Gly Asp Thr  ACC TTC GGC TTC TGG GCT GCC TAC CTG GCT GGC GGA GAC ACT 980 Thr Phe Gly Phe Trp Ala Ala Tyr Leu Ala Gly Gly Asp Thr
310 31S 320  310 31S 320
GTC TAC CTG GCC AAC TTC ACC CTG CCA GAC TCT GAG TTC CTG 1022 Val Tyr Leu Ala Asn Phe Thr Leu Pro Λβρ Ser Qlu Phe Leu  GTC TAC CTG GCC AAC TTC ACC CTG CCA GAC TCT GAG TTC CTG 1022 Val Tyr Leu Ala Asn Phe Thr Leu Pro Λβρ Ser Qlu Phe Leu
325 330 335  325 330 335
ΆΆ0 ATC TTT AAG CCG GAG GCG GCC TTC CTG CCC ΟΛΟ TOG GTG 1064 Lys lie Phe Lys Pro Glu Ala Ala Phe Leu Pro Glu Txp Val  ΆΆ0 ATC TTT AAG CCG GAG GCG GCC TTC CTG CCC ΟΛΟ TOG GTG 1064 Lys lie Phe Lys Pro Glu Ala Ala Phe Leu Pro Glu Txp Val
340 345 350  340 345 350
GGC ATT AAT GCA OAC TTG TCT CCA CTC TGG ACA TTG GCT AAG 1106 Gly lie Asn Ala Asp Leu Ser Pro Leu Trp Thr Leu Ala Lye  GGC ATT AAT GCA OAC TTG TCT CCA CTC TGG ACA TTG GCT AAG 1106 Gly lie Asn Ala Asp Leu Ser Pro Leu Trp Thr Leu Ala Lye
355 360  355 360
CCT T6A0AGCCAG GGAGACTTTC TGAAGTAGCC TGATCTTTCT 1149 CCT T6A0AGCCAG GGAGACTTTC TGAAGTAGCC TGATCTTTCT 1149
Pro Pro
365  365
AGAGCCAGCA GTACGTGGCT TCAGA 1174 ( 3) 配列番号 2 (SEQ ID N0:2)に関する情報 AGAGCCAGCA GTACGTGGCT TCAGA 1174 (3) Information on SEQ ID NO: 2 (SEQ ID N0: 2)
( i ) 配列の特徴：  (i) Sequence features:
(A) 長さ ： 1 4 2 3  (A) Length: 1 4 2 3
(B) 型：核酸  (B) Type: nucleic acid
(C) 鎖の数：ニ本鎮  (C) Number of chains: two main
(D) トポロジー ：直蛾状  (D) Topology: straight moth
(ii) 配列の種 ： cDNA to nRNA (ii) Sequence species: cDNA to nRNA
(A) 特 ffi . （ 1 ， 3 ) ガラク トシルトランスフェラーゼ. Ciii) ハイボセティカル ： N 0 (A), especially ffi (1, 3) galactosyltransferase Ciii) high Bose Tikal:.. N 0
(iv) アンチセンス ： N 0  (iv) Antisense: N 0
(X) 刊行物情報：  (X) Publication information:
( A) 著者： auro S. Sandrin  (A) Author: auro S. Sandrin
Paul L. Dabkowski  Paul L. Dabkowski
Margaret M. Kenning  Margaret M. Kenning
Bff ie Mouhtouris  Bff ie Mouhtouris
Ian F. C.Mckenzie  Ian F. C. Mckenzie
(B) 理名 ： Characterization of cDNA clones for porcine alfaCl.3)galactosyl transferase ： The enzyme generating the Gal alfaCl, 3)Cal epitope (B) Scientific name: Characterization of cDNA clones for porcine alfaCl. 3) galactosyl transferase: The enzyme generating the Gal alfaCl, 3) Cal epitope
CO 雜誌名： XENOTRANSPLANTATION CO Magazine Name: XENOTRANSPLANTATION
CD) 巻数： 1994;1  CD) Volume: 1994; 1
(F) 頁数： 81-88  (F) Pages: 81-88
(xi) 配列の記載： S E Q I D NO ： 2 ： CGGGGGCCAT CCCCGAOCGC ACCCAGCTTC TGCCGATCAG 6Α6ΆΆΆΑΤΑ 49(xi) Sequence description: SEQID NO: 2: CGGGGGCCAT CCCCGAOCGC ACCCAGCTTC TGCCGATCAG 6-6 49
ATG AAT GTC AAA GGA AGA GTG GTT CTG TCA ATG CTG CTT QTC 91 Met Asn Val Lys Gly Arg Val Val Leu Ser Met Leu Leu Val ATG AAT GTC AAA GGA AGA GTG GTT CTG TCA ATG CTG CTT QTC 91 Met Asn Val Lys Gly Arg Val Val Leu Ser Met Leu Leu Val
5 10  5 10
TCA ACT QTA ATG GTT GTG TTT TGG GAA TAC ATC AAC AQA AAC 133 Ser Thr Val Met Val Val Fhe Trp Qlu Tyr lie Asn Arg Asn  TCA ACT QTA ATG GTT GTG TTT TGG GAA TAC ATC AAC AQA AAC 133 Ser Thr Val Met Val Val Fhe Trp Qlu Tyr lie Asn Arg Asn
15 20 25  15 20 25
CCA GAA GTT GGC AGC AGT GCT CAG AGG GGC TGG TGG TTT CCG 175 Pro Glu Val Gly Ser Ser Ala Gin Arg Gly Trp Trp Phe Pro  CCA GAA GTT GGC AGC AGT GCT CAG AGG GGC TGG TGG TTT CCG 175 Pro Glu Val Gly Ser Ser Ala Gin Arg Gly Trp Trp Phe Pro
30 35 40  30 35 40
AGC TGG TTT AAC AAT GGG ACT CAC AGT TAC CAC GAA GAA GAA 217 Ser Trp Phe Asn Asn Gly Thr His Ser Tyr Hie Glu Glu Qlu  AGC TGG TTT AAC AAT GGG ACT CAC AGT TAC CAC GAA GAA GAA 217 Ser Trp Phe Asn Asn Gly Thr His Ser Tyr Hie Glu Glu Qlu
45 50 55  45 50 55
GAC GCT ATA GGC AAC GAA- AAG GAA CAA ACA AAA GAA GAC AAC 259 AB Ala He Gly Aan Glu Lye Glu Gin Arg Lye Glu Aap Asn  GAC GCT ATA GGC AAC GAA- AAG GAA CAA ACA AAA GAA GAC AAC 259 AB Ala He Gly Aan Glu Lye Glu Gin Arg Lye Glu Aap Asn
SO S5 70  SO S5 70
AGA GGA GAG CTT CCG CTA GTG GAC TGG TTT AAT CCT GAG AAA 301 Arg Gly Glu Leu Pro Leu Val Asp Trp Phe Aan Pro Glu Lys AGA GGA GAG CTT CCG CTA GTG GAC TGG TTT AAT CCT GAG AAA 301 Arg Gly Glu Leu Pro Leu Val Asp Trp Phe Aan Pro Glu Lys
75 BO  75 BO
CGC CCA GAG GTC GTQ ACC ATA ACC AQA TGG ΛΆΟ GCT CCA GTG 343 Arg Pro Glu Val Val Thr lie Thr Arg Trp Lys Ala Pro Val  CGC CCA GAG GTC GTQ ACC ATA ACC AQA TGG ΛΆΟ GCT CCA GTG 343 Arg Pro Glu Val Val Thr lie Thr Arg Trp Lys Ala Pro Val
85 90 ' 95  85 90 '95
C3TA TQG GAA GGC ACT TAC AAC AGA GCC GTC TTA GAT AAT TAT 385 Val Trp Glu Gly Thr Tyr Aen Arg Ala Val Leu Asp Asn Tyr  C3TA TQG GAA GGC ACT TAC AAC AGA GCC GTC TTA GAT AAT TAT 385 Val Trp Glu Gly Thr Tyr Aen Arg Ala Val Leu Asp Asn Tyr
100 105 no  100 105 no
TAT GCC AA% CAG AAA ATT ACC GTG GGC TTG ACG GTT TTT GCT 427 lyr Ala Lys Gin Lys lie Thr Val Gly Leu Thr Val Phe Ala  TAT GCC AA% CAG AAA ATT ACC GTG GGC TTG ACG GTT TTT GCT 427 lyr Ala Lys Gin Lys lie Thr Val Gly Leu Thr Val Phe Ala
US 120 125  US 120 125
GTC GGA AGA TAC ATT GAG CAT TAC TTG GAG GAG TTC TTA ATA 4 GTC GGA AGA TAC ATT GAG CAT TAC TTG GAG GAG TTC TTA ATA 4
Val Gly Arg Tyr lie Glu His Tyr Leu Glu Glu Phe Leu lie Val Gly Arg Tyr lie Glu His Tyr Leu Glu Glu Phe Leu lie
130 135 140  130 135 140
TCT GCA AAT ACA TAC TTC ATG GTT GGC CAC AAA GTC ATC TTT 511 Ser Ala Asn Thr Tyr Phe Met Val Gly His Lys Val lie Phe  TCT GCA AAT ACA TAC TTC ATG GTT GGC CAC AAA GTC ATC TTT 511 Ser Ala Asn Thr Tyr Phe Met Val Gly His Lys Val lie Phe
145 150  145 150
TAC ATC ATG GTG GAT GAT ATC TCC AGG ATG CCT TTG ATA GAG SS3 Tyr lie Met Val Asp Asp lie Ser Arg Met Pro Leu lie Glu  TAC ATC ATG GTG GAT GAT ATC TCC AGG ATG CCT TTG ATA GAG SS3 Tyr lie Met Val Asp Asp lie Ser Arg Met Pro Leu lie Glu
155 160 165 155 160 165
CTO GGT CCT CTG CQT TCC TTT AAA 6T0 TTT GAG ATC ΛΑΟ TCC 595 Leu Gly Pro Leu Arg Ser Phe Lys Val Fhe Qlu lie Lys Ser  CTO GGT CCT CTG CQT TCC TTT AAA 6T0 TTT GAG ATC ΛΑΟ TCC 595 Leu Gly Pro Leu Arg Ser Phe Lys Val Fhe Qlu lie Lys Ser
170 175 180 GAG AAQ ASG TGG CAA. SAC ATC AGC ATG AT6 C6C ATG AAG ACC 637 Glu Lys Arg Trp Gin Asp lie Ser Met Met Arg Met Lys Thr 170 175 180 GAG AAQ ASG TGG CAA. SAC ATC AGC ATG AT6 C6C ATG AAG ACC 637 Glu Lys Arg Trp Gin Asplie Ser Met Met Arg Met Lys Thr
1B5 190 195  1B5 190 195
ATC GGQ GAG CAC ATC CTG GCC CAC ATC CAG CAC GAfi GTG GAC 679 He Gly Glu His lie Leu Ala His lie Gin His Glu Val Asp  ATC GGQ GAG CAC ATC CTG GCC CAC ATC CAG CAC GAfi GTG GAC 679 He Gly Glu His lie Leu Ala His lie Gin His Glu Val Asp
200 205 210  200 205 210
TTC CTC TTC TGC ATT GAC GTG GAT CAQ GTC TTC CAA AAC AAC 721 Phe Leu Phe Cys lie Asp Val Asp Gin Val Phe Gin Aen Asn  TTC CTC TTC TGC ATT GAC GTG GAT CAQ GTC TTC CAA AAC AAC 721 Phe Leu Phe Cys lie Asp Val Asp Gin Val Phe Gin Aen Asn
215 220  215 220
TTT GGG GTG GAG ACC CTG GGC CAQ TCG GTG GCT CAG CTA. CAG 763 Phe Qly Val Olu Thr Leu Gly Gin Ser Val Ala Gin Leu Gin  TTT GGG GTG GAG ACC CTG GGC CAQ TCG GTG GCT CAG CTA. CAG 763 Phe Qly Val Olu Thr Leu Gly Gin Ser Val Ala Gin Leu Gin
225 230 235 225 230 235
GCC TGG TGQ TAC AAQ GCA CAT CCT GAC GAG TTC ACC TAC GAG 805 Ala Trp Trp Tyr Lys Ala His Pro Asp Glu Phe Thr Tyr Glu  GCC TGG TGQ TAC AAQ GCA CAT CCT GAC GAG TTC ACC TAC GAG 805 Ala Trp Trp Tyr Lys Ala His Pro Asp Glu Phe Thr Tyr Glu
240 245 250  240 245 250
AGG CGG AAG GAG TCC GCA GCC TAC ATT CCG TTT GGC CAG GGG 847 Arg Arg Lys Glu Ser Ala Ala Tyr lie Pro Phe Gly Gin Gly  AGG CGG AAG GAG TCC GCA GCC TAC ATT CCG TTT GGC CAG GGG 847 Arg Arg Lys Glu Ser Ala Ala Tyr lie Pro Phe Gly Gin Gly
255 260 265  255 260 265
GAT TTT TAT TAC CAC GCA GCC ATT TTT GGG GGA ACA CCC ACT 889 Asp Phe Tyr Tyr His Ala Ala lie Phe Gly Gly Thr Pro Thr GAT TTT TAT TAC CAC GCA GCC ATT TTT GGG GGA ACA CCC ACT 889 Asp Phe Tyr Tyr His Ala Ala lie Phe Gly Gly Thr Pro Thr
270 275 280  270 275 280
CAG GTT CTA AAC ATC ACT CAG GAG TGC TTC AAG GGA ATC CTC 931 CAG GTT CTA AAC ATC ACT CAG GAG TGC TTC AAG GGA ATC CTC 931
Gin Val Leu Asn lie Thr Gin Qlu Cys P^e Lys Gly lie Leu Gin Val Leu Asn lie Thr Gin Qlu Cys P ^ e Lys Gly lie Leu
265 290  265 290
CAG GAC AAG GAA ΑλΤ QAC ATA GAA GCC GAG TQG CAT GAT GAA 973 Gin Asp - Lye Glv Asn Asp lie Olu Ala Glu Trp His Asp Glu  CAG GAC AAG GAA ΑλΤ QAC ATA GAA GCC GAG TQG CAT GAT GAA 973 Gin Asp-Lye Glv Asn Asplie Olu Ala Glu Trp His Asp Glu
295 300 305 295 300 305
AGC CAT CTA AAC AAQ TAT TTC CTT CTC AAC ΆΆΆ CCC ACT AAA 1015 Ser His Leu Asn Lys Tyr Phe Leu Leu Asn Lys Pro Thr Lys  AGC CAT CTA AAC AAQ TAT TTC CTT CTC AAC ΆΆΆ CCC ACT AAA 1015 Ser His Leu Asn Lys Tyr Phe Leu Leu Asn Lys Pro Thr Lys
310 315 320  310 315 320
ATC TTA TCC CCA GAA TAC TGC TGG GAT TAT CAT ATA GGC ATG 1057 lie Leu Ser Pro Glu Tyr Cys Trp Aep Tyr His lie Gly Met  ATC TTA TCC CCA GAA TAC TGC TGG GAT TAT CAT ATA GGC ATG 1057 lie Leu Ser Pro Glu Tyr Cys Trp Aep Tyr His lie Gly Met
325 330 335 TCT GTG GAT ATT AGG ATT GTC AAG ATA GCT TGG CAG AAA AAA. 1039 Ser Val Asp lie Arg lie Val Lys lie Ala Tip Gin Lye Lys 325 330 335 TCT GTG GAT ATT AGG ATT GTC AAG ATA GCT TGG CAG AAA AAA.1039 Ser Val Asp lie Arg lie Val Lys lie Ala Tip Gin Lye Lys
340 345 350  340 345 350
GAG TAT .AAT TTG GTT ASA AAT AAC ATC TGACTTTAAA 113 G 61u Tyr Asn Lau Val Arg A9zi Aen lie  GAG TAT .AAT TTG GTT ASA AAT AAC ATC TGACTTTAAA 113 G 61u Tyr Asn Lau Val Arg A9zi Aen lie
355  355
TTGTGCCAQC AGTTTTCTGA ATTT6AAAGA GTATTACTCT GGCTACTTCC 118G TTGTGCCAQC AGTTTTCTGA ATTT6AAAGA GTATTACTCT GGCTACTTCC 118G
TCAGAGAAGT AGCACTTAAT TTTAACTTTT ΑΑΑΆΑΆΛΤΑΟ TAACAAAATA 1236TCAGAGAAGT AGCACTTAAT TTTAACTTTT ΑΑΑΆΑΆΛΤΑΟ TAACAAAATA 1236
CCAACftCAGT AAGTACATAT TATTCTTCCT TGCAACTTTG AGCCTTGTCA 1286CCAACftCAGT AAGTACATAT TATTCTTCCT TGCAACTTTG AGCCTTGTCA 1286
AATGGGAGAA TGACTCTGTA GTAATCAGAT GTAAATTCCC AATGATTTCT 1336AATGGGAGAA TGACTCTGTA GTAATCAGAT GTAAATTCCC AATGATTTCT 1336
TATCTGCGGA ATTCCAGCTG AGCGCCGGTC GCTACCATTA CCAGTTGGTC 138 STATCTGCGGA ATTCCAGCTG AGCGCCGGTC GCTACCATTA CCAGTTGGTC 138 S
TGGTGTCGAC GACTCCTGGA GCCCGTCAGT ATCGGCG 1423 TGGTGTCGAC GACTCCTGGA GCCCGTCAGT ATCGGCG 1423
(4)配列番号 3 (SEQ ID N0:3)に関する情箝 (4) Information about SEQ ID NO: 3 (SEQ ID N0: 3)
( i )配列の特敏：  (i) Array sensitivity:
(A)長さ ： S 8  (A) Length: S8
(B)型：核酸  Type (B): nucleic acid
(C) 鎮の數： 1本钹  (C) Number of towns: 1
(D) トポロジー ：直蛾伏  (D) Topology: Namobushi
(ϋ) 配列の種類： その他の核酸  (ϋ) Sequence type: Other nucleic acids
(iii) ハイボセティ カル ： N 0  (iii) Hibosetical: N 0
(iv〉 アンチセンス ： No  (iv) Antisense: No
(vi)配列の記載： SEQ I D NO： 3 ；  (vi) Sequence description: SEQ ID NO: 3;
GTACCTTCCT TTCCTCTGCT GAGCCCTGCC TCCTTAGG 38 GTACCTTCCT TTCCTCTGCT GAGCCCTGCC TCCTTAGG 38
(5) 配列番号 4 (SEQ ID N0:4)に関する情報 (5) Information on SEQ ID NO: 4 (SEQ ID N0: 4)
( i ) 配列の特徵：  (i) Array features:
(A) 長さ ： 33  (A) Length: 33
(B) 型：核酸  (B) Type: nucleic acid
(C) 鎖の数： 1本鎮  (C) Number of chains: 1 strand
(D) トポロジー ：直縝状  (D) Topology: straight
Cii)配列の種類： その他の核酸  Cii) Sequence type: Other nucleic acids
(iii) ハイボセティカル ： No  (iii) Hibothetical: No
(iv) アンチセンス ： N 0  (iv) Antisense: N 0
(vi) 配列の記載： SEQ I D NO： 4 ：  (vi) Sequence description: SEQ ID NO: 4:
GGGGTAAGCA GATCTCTTGA GTTCAAAGTC AGC 33 GGGGTAAGCA GATCTCTTGA GTTCAAAGTC AGC 33
(6)配列番号 5 (SEQ ID N0:5)に関する情報 (6) Information on SEQ ID NO: 5 (SEQ ID N0: 5)
( i ) 配列の特徴： 96/28967 一 4 1一 (i) Sequence features: 96/28967 1 4 1 1
(A)長さ ： 27 (A) Length: 27
(B)型 ：核酸  (B) type: nucleic acid
(C) 鎖の数： 1本蛾  (C) Number of chains: 1 moth
(D) トポロジー ：直镇状  (D) Topology: straight
(ϋ) 配列の種類： その他の核酸  (ϋ) Sequence type: Other nucleic acids
Ciii) ハイポセティ カル ： N 0  Ciii) Hypothetical: N 0
(iv) アンチセンス ： No  (iv) Antisense: No
(vi)配列の記載： SEQ I D NO： 5  (vi) Sequence description: SEQ ID NO: 5
CTTTAATTCC AGTCGACTGG GGTAAGC 27 CTTTAATTCC AGTCGACTGG GGTAAGC 27
(7) 配列番号 6 (SBQ ID N0:6)に関する情報 (7) Information on SEQ ID NO: 6 (SBQ ID N0: 6)
( i ) 配列の特徴：  (i) Sequence features:
(A)長さ ： 30  (A) Length: 30
CB)型 ：核酸  CB) type: nucleic acid
(C)鎖の数： 1本鎖  (C) Number of chains: 1 strand
(D) トポロジー ：直縝状  (D) Topology: straight
(ϋ) 配列の種頷： その他の核酸  (ϋ) Sequence nod: other nucleic acids
(iii) ハイボセティ カル ： No  (iii) Hibosetical: No
(iv) アンチセンス ： N 0  (iv) Antisense: N 0
(vi) 列の記裁： S E-Q I D NO： 6  (vi) Column notation: S E-Q I D NO: 6
GTCAAGGGAA AAGTAATCCT GTTGATGCTG 30 GTCAAGGGAA AAGTAATCCT GTTGATGCTG 30
(8)配列番号 7 (SEQ ID N0:7)に関する情報 (8) Information on SEQ ID NO: 7 (SEQ ID N0: 7)
( i )配列の特徴：  (i) Array features:
(A) 長さ ： S 4  (A) Length: S 4
(B) 型 ：核酸  (B) Type: nucleic acid
(C) 鎖の数： I本鎖 (D) トポロジー ：直镇状 (C) Number of chains: I-strand (D) Topology: straight
(ϋ) 配列の種類：その他の核酸  (ϋ) Sequence type: Other nucleic acids
(ώ) ハイボセティカル ： No  (ώ) Hibothetical: No
(iv) アンチセンス ： N o  (iv) Antisense: No
(vi)配列の記載： SEQ I D NO : 7 :  (vi) Sequence description: SEQ ID NO: 7:
CCAGCTTGGG AACCACCAGT CCTTCTGCCA TCTG 34 CCAGCTTGGG AACCACCAGT CCTTCTGCCA TCTG 34
(9) 配列番号 8 (SEQ ID N0:8)に関する情報 (9) Information on SEQ ID NO: 8 (SEQ ID N0: 8)
( i ) 配列の特徴：  (i) Sequence features:
(A) 長さ ： 26  (A) Length: 26
(B)型 ：核酸  (B) type: nucleic acid
(C) 镇の数： 1本鑌  (C) Number of 1: 1 鑌
CD) トポロジー ：直鎮状  CD) Topology: Direct letter
Cii) 配列の種類 ·· その他の核酸  Cii) Sequence typeOther nucleic acids
(iii) ハイポセティカル ： N 0  (iii) Hypothetical: N 0
(iv) アンチセンス ： No  (iv) Antisense: No
Cvi) 配列の記載： S E Q I D NO： 8 ：  Cvi) Sequence description: SEQUIDNO: 8:
ACGAAAAGCG GACTGTCGAC CTCCCA 26 ACGAAAAGCG GACTGTCGAC CTCCCA 26
( 1 0) 配列番号 9 (SEQ ID N0:9)に閭する情報 (1 0) Information provided in SEQ ID N0: 9
( i) 配列の特徴：  (i) Sequence features:
(A) 長さ ： 33  (A) Length: 33
(B) 型：核酸  (B) Type: nucleic acid
(C) 鎖の数： 1本鎖  (C) Number of chains: single strand
(D) トポロジー ：直鎖状  (D) Topology: linear
(ii) 配列の種類： その他の核酸  (ii) Sequence type: Other nucleic acids
(iS) ハイボセティカル ： No  (iS) Hibo Setical: No
(iv) アンチセンス ： N 0  (iv) Antisense: N 0
(vi) 配列の記載： SEQ I D NO： 9 ：  (vi) Sequence description: SEQ ID NO: 9:
CCCTCCCTCT CAAGGCTTAG CCAATCTCCA GAG 38 CCCTCCCTCT CAAGGCTTAG CCAATCTCCA GAG 38
( 1 1 ) 配列番号 1 0 (SEQ ID N0:10) に関する情報 (11) Information on SEQ ID NO: 10 (SEQ ID N0: 10)
( i ) 配列の特徼：  (i) Special array:
(A) 長さ ： 29  (A) Length: 29
(B) 型 ： 核酸  (B) Type: nucleic acid
(C) 鎖の数： 1本鎮  (C) Number of chains: 1 strand
(D) トポロジー ：直鎮状  (D) Topology: direct letter
(ii) 配列の種類： その他の核酸  (ii) Sequence type: Other nucleic acids
(iii) ハイボセティカル ： No  (iii) Hibothetical: No
(iv) アンチセンス ： No  (iv) Antisense: No
(vi) 配列の記載： SEQ I D NO ： 1 0 ：  (vi) Sequence description: SEQ IDNO: 10:
AGAGCTGACG ATCGCTCCGG AGCCACATC 29 ) 配列番号 1 1 (SEQ ID N0:11) に関する情報 AGAGCTGACG ATCGCTCCGG AGCCACATC 29 ) Information on SEQ ID NO: 1 1 (SEQ ID N0: 11)
( i ) 配列の特徴：  (i) Sequence features:
(A) 長さ ： 32  (A) Length: 32
(B) 型：核酸  (B) Type: nucleic acid
(C) 鎮の數： 1本镇  (C) Number of towns: 1
CD) トポロジー ：直錤状  CD) Topology: Straight
(ii) 配列の種類： その他の核酸  (ii) Sequence type: Other nucleic acids
(iii) ハイボセティ カル ： N 0  (iii) Hibosetical: N 0
(iv) アンチセンス ： No  (iv) Antisense: No
(vi) 配列の記載： SEQ I D NO : 1 1 :  (vi) Sequence description: SEQ ID NO: 1 1:
CCACTCTGGA CATTGGCTAA GCCTTGACAG CC 32  CCACTCTGGA CATTGGCTAA GCCTTGACAG CC 32

Claims

if求の $ε囲 $ ε enclosure of if request
1. 高等 S長類の α ( 1 , 2) フコシルトランスフェラーゼをコードする DNA 配列を含む DN Α構築物が導入され、 H型抗原を発現するように形質転換されて いることを特徴とする非蕙長哺乳類の形質転換動物。 1. A non-human length characterized in that a DNΑ construct containing a DNA sequence encoding a higher S-long α (1,2) fucosyltransferase has been introduced and has been transformed to express an H-type antigen. Transgenic mammal.
2. 高等霊長類のな （ 1 , 2) フコシルトランスフ ラーゼをコードする DNA 配列を含む第 1の DN A桷築物と、 非霊長哺乳類のな （し 3) ガラク トシルト ランスフェラーゼの遣伝子に対してアンチセンスである DN A配列を含む第 2の DNA構築物とが導入され、 高等霊長類の H型抗原を発現する一方、 非 S長哺乳 類の G型抗原を低減するように形質転換されていることを特徴とする非霊長哺乳 類の形質転換動物。  2. The first DNA construct containing a DNA sequence encoding the (1,2) fucosyltransferase of higher primates and the gene for galactosyltransferase of non-primate mammals (3) A second DNA construct containing the antisense DNA sequence was introduced and transformed to express higher primate H-type antigens while reducing non-S-length mammalian G-type antigens. A transgenic non-primate mammal, characterized in that:
3. 非霊長哺乳類の α ( 1 , 3) ガラク トシルトランスフユラーゼ逭伝子の一部 に、 a ( 1. 2) フコシルトランスフェラーゼをコードする DN A配列を含む D NA構築物か相同組換えにより挿入され、 非霊長哺乳類の G型抗原を発現するこ となく高等靈長類の H型抗原を発現するように形質転換されていることを特徴と する非霊長哺乳類の形質転換動物。  3. Insertion of a DNA construct containing a (1.2) fucosyltransferase into a DNA construct containing a (1.2) fucosyltransferase by homologous recombination into a part of the α (1,3) galactosyltransferase 逭 gene A non-primate mammal transformed to express a higher primate H-type antigen without expressing the non-primate mammal's G-type antigen.
4. 前記非霊長哺乳類の形質転換動物がブタであることを特徴とする請求項 1か ら 3のいずれかに記載の形質転換動物。  4. The transgenic animal according to any one of claims 1 to 3, wherein the transgenic non-primate mammal is a pig.
5. 非霊長哺乳類の a ( 1 , 3) ガラク トシルトランスフ ラーゼ ¾伝子の一部 に、 a ( 1 , 2) フコシル トランスフェラーゼをコードする DNA配列を含む D NA構築物が相同組換えにより挿入され、 非霊長哺乳類の G型抗原を発現するこ となく高等 II長類の H型抗原を発現するように形質転換された非霊長哺乳類の形 質転換動物から得られた移植用材料。  5. A DNA construct encoding a (1,2) fucosyltransferase is inserted into a portion of a (1,3) galactosyltransferase gene of a non-primate mammal by homologous recombination, Transplant material obtained from a transgenic non-primate mammal that has been transformed to express a higher II-type H antigen without expressing the G-type antigen of the non-primate mammal.
6. 非靈長哺乳類のな ( 1 , 3) ガラク トシル卜ランスフ ラーゼ遺伝子の一部 に. な （ 1. 2) フコシル トランスフ ラーゼをコー ドする DNA配列を含む D NA構築物を相同組換えにより挿入し、 非靈長哺乳類の G型抗原を発現すること なく高等霊長類の H型抗原を発現するように形質転換した非霊長哺乳類の形質転 換動物の作出方法。  6. Non-primate mammalian non-primate (1,3) DNA construct containing DNA sequence coding for fucosyltransferase was inserted by homologous recombination into a part of the (1,2) galactosyltransferase gene. And a method for producing a transgenic non-primate mammal transformed to express the H-type antigen of a higher primate without expressing the G-type antigen of the non-primate mammal.
7. 非霊長哺乳類の組織を高等置長類に移植する際の， 高等靈長類の自然抗体と 非 S長哺乳類の抗原とによる拒絶反応を回避する方法であって， 非霊長哺乳類のな （I, 3) ガラク トシル トランスフヱラ一ゼ逭伝子の一部に， ( 1, 2) フコシル トランスフ ラーゼをコー ドする DNA配列を含む DNA 搆築物を相同組換えにより挿入して， 非霊長哺乳類の Ggl抗原を発現することな く高等霊長類の H型抗原を発現するように形質転換した非霊長喃乳類を作出する 工程と， 7. When transplanting non-primate mammal tissues to higher elongates, A method for avoiding rejection by antigens of non-S long mammals, in which (1,2) fucosyltransferase is added to a part of (I, 3) galactosyltransferase gene in non-primate mammals. A non-primate primate transformed to express a higher primate H-type antigen without expressing the non-primate Ggl antigen by inserting a DNA construct containing the coding DNA sequence by homologous recombination. The process of producing milk,
この工程により得られた前記非霊長哺乳類の組截を高等 S長類に移植する工程 を含んだ方法。  Transplanting the non-primate mammal section obtained in this step into a higher S long-term mammal.
PCT/JP1996/000703 1995-03-17 1996-03-18 Transgenic non-primatal mammals wherein serotypes of higher primates have been expressed by foreign gene transfer and method of creating the same WO1996028967A1 (en)

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AU49562/96A AU4956296A (en) 1995-03-17 1996-03-18 Transgenic non-primatal mammals wherein serotypes of higher primates have been expressed by foreign gene transfer and method of creating the same
PCT/JP1996/000703 WO1996028967A1 (en) 1995-03-17 1996-03-18 Transgenic non-primatal mammals wherein serotypes of higher primates have been expressed by foreign gene transfer and method of creating the same
JP52828796A JP3809189B2 (en) 1995-03-17 1996-03-18 Non-primate mammal transformed animal expressing higher primate antigen type by introduction of foreign gene and method for producing the same

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JPPCT/JP95/00488 1995-03-17
PCT/JP1995/000488 WO1996028966A1 (en) 1995-03-17 1995-03-17 Transformant of nonprimatial mammal having serotype of advanced primate expressed by foreign gene transduction and process for producing the same
PCT/JP1996/000703 WO1996028967A1 (en) 1995-03-17 1996-03-18 Transgenic non-primatal mammals wherein serotypes of higher primates have been expressed by foreign gene transfer and method of creating the same

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EP0769902A4 (en) * 1994-06-15 2001-02-28 Alexion Pharma Inc Methods for reducing hyperacute rejection of xenografts
US6965022B2 (en) 1997-05-20 2005-11-15 The United States Of America As Represented By The Secretary Of Agriculture Methods to identify swine genetically resistant to F18 E. coli associated diseases
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WO2002074948A2 (en) * 2001-03-21 2002-09-26 Geron Corporation Animal tissue with carbohydrate antigens compatible for human transplantation
US7795493B2 (en) 2002-08-21 2010-09-14 Revivicor, Inc. Porcine animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US8106251B2 (en) 2002-08-21 2012-01-31 Revivicor, Inc. Tissue products derived from porcine animals lacking any expression of functional alpha 1,3 galactosyltransferase
US10130737B2 (en) 2002-08-21 2018-11-20 Revivicor, Inc. Tissue products derived from animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US10912863B2 (en) 2002-08-21 2021-02-09 Revivicor, Inc. Tissue products derived from animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US11172658B2 (en) 2002-08-21 2021-11-16 Revivicor, Inc. Porcine animals lacking expression of functional alpha 1, 3 galactosyltransferase
US7560538B2 (en) 2003-11-05 2009-07-14 University Of Pittsburgh Porcine isogloboside 3 synthase protein, cDNA, genomic organization, and regulatory region

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