Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
  • A01K67/027—New breeds of vertebrates
  • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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)

Definitions

• the present invention relates to the field of genetics and methods of altering the genetic material of an organism.
• the invention has a wide variety of applications, for example, in animal and plant breeding, including transgenosis, in the field of epigenetics, which deals with the variation in gene expression during differentiation and development of eucaryotic organisms, and in the understanding and treatment of genetic diseases.
• mutant hamster gene which codes for an altered dihydrofolate reductase, as a selectable marker offering methotrexate resistance, allows the introduction and amplification of a broad range of genetic elements into a variety of cell lines, Wigler, M., et al, Proc. Natl. Acad. Sci.
• this technique does not result in the production of a mature organism much less one that could exhibit a phenotypic alteration.
• Mosaic mice have been constructed by the injection of teratocarcinoma cells into the blastocysts of developing mice.
• a mosaic mouse has patches of different, mutually exclusive genotypes and/or phenotypes. Additionally, the possibility of germ-line transmission with mosaic mice is reduced due to the fact that teratoma cells of XX chromosomal constitution cannot make sperm in mice that develop as males.
• mice developed from embryos microinjected with the TK gene incorporated into a chimeric SV40 viral vehicle. These mice, however, did not express the TK gene product. In these
• zygote and the embryo and mature organism which result therefrom is obtained by the placing or insertion of exogenous genetic material. Additionally, the inclusion of the exogenous genetic material in the zygote will result in a phenotypic expression of the exogenous genetic material.
• the genotype of the exogenous genetic material is expressed upon the cellular division of the zygote. However, the phenotypic expression, e.g., the production of a protein product or products of the exogenous genetic material or alteration of the zygote ' s or organism's development during which the particular exogenous genetic material is active.
• Alteration of the expression of a phenotype includes an enhancement or dimishment in the expression of a phenotype or an alteration in the promotion and/or control of a phenotype including the addition of a new promotor and/or controller or supplementation of an existing promotor and/or controller of a phenotype.
• the present invention has application in the genetic transformation of multicellular eucaryotic organisms which undergo syngamy, i.e., sexual reproduction by the union of gamete cells.
• Examples of such organisms include amphibians, reptiles, birds, mammals, bony fishes, cartilaginous fishes, cyclostomes, arthropods, insects, mollusks, thallophytes, embryophytes including gymnosperms and angiosperms.
• Preferred organisms include mammals, birds, fishes, gymnosperms and angiosperms.
• the invention is particula rly usefu l in the breeding of plants and animals , especially ones of ag ricultu ral values , to obtain species having a genetic makeup which results in a plant or animal having more desi rable cha racteristics .
• the sou rce of exogenous genetic material can be from animals or plants , synthetic equivalents of naturally occurring genetic material or totally new synthetical ly produced genetic material and from the same or a different species of the zygote being transformed
• the invention can be used to modify a species or create a new species . Modification of a species is obtained when the genotype of the species whose zygote is being genetically transformed .
• a new species is obtained when the genotype of the exogenous genetic material occurs in another species and does not naturally occur in the species of the zygote being genetically transformed .
• increased growth rate and the efficiency of feed utilization can be obtained by genetic transformation of animals used to produce meat.
• the genes relating to growth rate and feed utilization can be transferred from a buffalo into beef cattle which would create a new species .
• Dairy animals can undergo an increase in milk production and efficiency of feed utilization by transferring exogenous genetic material from species or breeds of the same species which have either or both traits .
• the quality and flavor of meat, for example, lamb can also be enhanced in a similar manner.
• the invention can be used as an n_ vivo analysis of gene expression during differentiation and in the elimination or dimunition of genetic diseases , e. g. , hemophilia, Tay-Sachs disease, phenylketonuria, homocystinurea, galactosemia, thalassemia and sickle cell anemia .
• genetic diseases e. g. , hemophilia, Tay-Sachs disease, phenylketonuria, homocystinurea, galactosemia, thalassemia and sickle cell anemia .
• Figu re 1 is a photomicrograph of microinjectio ⁇ of rabbit fs-globin gene into the male pronucleus of a mouse zygote.
• Figure 2 is a photograph of ouchterlongy agarose gel diffusion reactions of antirabbit hemoglobin antiserum of Well A and normal nonimmunized mouse serum of Well B with a LT/Sv mouse contained in Wells 1, rabbit hemoglobin preparation contained in Wells 2 and a hemoglobin preparation from mice developed from microinjected zygotes contained in Wells 3.
• Figure 3 is a photograph of ouchterlongy agarose gel diffusion reactions of antirabbit hemoglobin antiserum contained in Well A with dissociated rabbit hemoglobin preparation contained in Wells 1, 4 and 6, hemoglobin from a normal F, hybrid C57BL/6J X LT/Sv mouse contained in Well 5, dissociated rabbit globin chains contained in Well 2 and a hemoglobin preparation from mice developed from the ⁇ -globin gene microinjected zygotes contained in Well 3.
• Figure 4 is a photograph of ouchterlongy agarose gel diffusion reactions conducted in a manner identical to those of Fig. 2 except Well B contains antirabbit hemoglobin antiserum absorbed by apssage through a gel immobilized rabbit hemoglobin preparation.
• Zygote is a diploid cell having the potential for development into a complte organism.
• the zygote can result from parthenogensis, nuclear transplantation, the merger of two gametes by artificial or natural fertilization or any other method which creates a diploid cell having the potential for development into a complete organism.
• the origin of the zygote can be from either the plant or animal kingdom.
• Parthenogenesis is any technique that allows for the development of a female or male gamete into a cell and its development into an organism which technique is different from the natural development of female and male gametes .
• Genetic material is a material containing any DNA sequence or sequences either purified or in a native state such as a fragment of a chromosome or a whole chromosome, either natu rally occurring or synthetically or partially synthetically prepared DNA sequences, DNA sequences which constitute a gene or genes and gene chimeras, e.g . , created by ligation of different DNA sequences. Genetic material does not include DNA sequences incorporated in or carried by a plasmid, virus or phage.
• Exogenous genetic material is a genetic material not obtained from or does not naturally form a part of the specific germ cells or gametes which form the particular zygote which is being genetically transformed .
• DNA sequence is a linear sequence comprised of any combination of the four DNA monomers, i . e. , nucleotides of adenine, quanine, cytosine and thymine, which codes for genetic information , such as a code for an am ⁇ no acid, a promoter, a control or a gene product.
• a specific DNA sequence is one which has a known specific function, e.g. , codes for a particular polypeptide, a particular genetic trait or affects the expression of a particular phenotype.
• Gene is the smallest, independently functional unit of genetic matieral which codes for a protein
• CM PI product or controls or affects transcription and comprises at least one DNA sequence.
• Geneotype is the genetic constitution of an organism.
• Phenotype is a collection of morphological, physiological and biochemical traits possessed by a cell or organism that results from the interaction of the genotype and the environment.
• Phenotypic expression is the expression of the code of a DNA sequence or sequences which results in the production of a product, e.g., a polypeptide or protein, or alters the expression of the zygote's or the organism's natural phenotype.
• Chrosome is a fiber or threadlike structure which is completely or partically composed of genetic nucleic acid.
• Chrosomal material is comprised of portions of or whole native chromosomes, isolated metaphase chromosomes (Wray, W. , et al, Exp. Cell Res. , 59:469- 478 (1970)) or artificial chromosomes composed of native or modified histone or protamine chromosomal core complexes and DNA. Artificial chromosomes can be prepared, for example, by removing a portion of the naturally occurring DNA sequences of a native chromosome and replacing it with new DNA sequences.
• the exogenous genetic material can be added to any nucleic genetic material which ultimately forms a part of the nucleus of the zygote, including the zygote nucleus.
• the nucleic genetic material must be in a physical state which enables it to takeup the exogenous genetic material.
• the exogenous genetic material can be placed in the nucleus of a primordial germ cell which is deplo ⁇ d, e.g., a spermatogonium or oogon ⁇ um.
• the primordial germ cell is then allowed to mature to a gamete which is then united with another gamete or source of a haploid set of chromosomes to form a zygote.
• the exogenous genetic material can be placed in the nucleus of one of the gametes, e.g., a mature sperm, egg or polar body, which forms a part of the zygote.
• the exogenous genetic material if the exogenous genetic material is placed in the mature sperm prior to fertilization, the sperm cell should be induced to undergo decondensat ⁇ on. Otherwise, the chromosomal complement of the sperm cell is too dense to allow the addition of any material to its nucleus.
• Techniques for the decondensation are known in the art. Examples of such techniques can be found in Mahi, D.A., et al, J. Rep rod. Fert., 44:293-296 (1975); Hendricks, D.M.
• the exogenos genetic material can be placed in the nucleus of the mature egg. It is preferred that the egg be in a fertilized or activated, by parthenogenesis, state. After the addition of the exogenous genetic material, then a complementary haploid set of chromosomes, e.g., a sperm cell or polar body, is added to enable the formation of zygote. When a polar body is used to diploidize a haploid egg, the exogenous genetic material can be placed in the polar body.
• a complementary haploid set of chromosomes e.g., a sperm cell or polar body
• the exogenous genetic material be placed in either the male or female pronucleus of the zygote.
• the male pronucleus is the preferred site for addition of the exogenous genetic material.
• the exogenous genetic material be chromosomal material, more preferably that the chromosomal material be comprised of histone chromosomal core complexes and DNA, which can be artificial chromosomal material reconstituted from DNA sequences of native or modified histone chromosomal core complexes.
• the exogenous genetic material be added to the male DNA complement, or a DNA complement other than the DNA complement of the female pronucleus, of the zygote prior to its being processed by the ovum nucleus or the zygote female pronucleus. It is thought that the ovum nucleus or female pronucleus release molecules which affect the male DNA complement, perhaps by replacing the protamines of the male DNA with histones , thereby facilitating the combination of the female and male DNA complements to form the diploid zygote.
• the processing of a DNA complement by the female pronucleus of the zygote refers to a biochemical effect the female pronucleus has on the DNA complement prior to the physical contact between the pronuciei or between the female pronucleus and the particular DNA complement.
• the exogenous genetic material be added to the male complement of DNA or any other complement of DNA prior to its being affected by the female pronucleus.
• the exogenous genetic material is added to the early male pronucleus, as soon as possible after the formation of the male pronucleus, which is when the male and female pronuciei are well separated and both are located close to the cell membrane.
• the exogenous genetic material could be added to the nucleus of the sperm after it has been induced to undergo decondensation .
• Sperm containing the exogenous genetic material could then be added to the ovum or the decondensed sperm could be added to the ovum with the exogenous genetic material being added as soon as possible thereafter.
• the exogenous material be added to that female DNA complement shortly after its placement within the ovum or prior to its placement in the ovum in order to allow that DNA complement to be processed by the female pronucleus .
• a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism.
• the zygote will be comprised of an egg containing a nucleus formed , either naturally or artifically, by the fusion of two haploid nuclei from a gamete or gametes .
• the gamete nuclei must be ones which are natu rally compatible, i . e. , ones wh ich result in a viable zygote capable of undergoing differentiation and developing into a functioning organism .
• a euploid zygote is preferred.
• the number of chromosomes should not vary by more than one with respect to the euploid number of the organ ism from which either gamete originated .
• physical ones also govern the amount of exogenous genetic material which can be added to the nucleus of the zygote or to the genetic material which forms a part of the zygote nucleus . If no genetic material is removed, then the amount of exogenous genetic material which can be added is limited by the amount which will be absorped without being physically disruptive. Generally, the volume of exogenous genetic material inserted will not exceed about 10 picoliters.
• exogenous genetic material can be added if it is exchanged for existing genes . Then , to a certain extent, the amount added is unlimited as long as the number of existing genes being removed is equal to the number inserted. However, the physical effects of deletion and addition must not be so great as to physically destroy the viability of the zygote.
• the biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one in the art, because the genetic material , including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism .
• the amount inserted will not be greater than one chromosome and preferably will be less than one chromosome.
• the number of copies of the DNA sequences which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoret ⁇ ically only one copy is required; however, generally, numerous copies are utilized, for example, 1 ,000-20,000 copies of a gene, in order to insure that one copy is functional .
• the particular composition or form of the exogenous genetic material is not critical . If a particular trait is desired to be incorporated into an organism, then the genetic material needs to contain the DNA sequence or sequences, or gene or loci, which code for the trait. Thus, whether the exogenous genetic material is a whole ch romosome, a portion of a chromosome, unpurified native DNA or synthetic or semi- synthetic DNA or chromosomal complexes is dependent upon the particular trait or traits sought to be incorporated into the zygote and ease of obtaining that trait.
• control sequences of the genetic material responsible for controlling the gene(s) coding for the trait particularly when the genotype of the zygote contains no similar gene whose control region could be used to regulate the gene or when greater genetic activity is desired.
• a control region e.g., an artifical one spliced to the exogenous gene, which will activate the gene when the organism is exposed to a different stimulus other than the natural stimulus of the gene.
• a growth hormone gene inserted into a bovine zygote could be attached to a control gene which is responsive to a molecule which could be added to the feed of the animal. Thus, each time the animal ingested the molecule, the growth hormone gene would be activated.
• V.'IFO zygotes for example, mammals, it will be necessary to complete the development of the zygote _m vivo by implanting it into a foster mother. It is preferred that the implantation occu rs at the moruia or blastocyst stage of development. Any tech nique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, n uclear membrane or other existing cellular or genetic structures . The exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection . Microinjection of cells and cellular structures is known and is used in the art.
• Hha I restriction endonuclease (Plasmid pCRI has numerous Hha I sites while the ⁇ -globin has none).
• the 6200 base pair ⁇ -globin gene DNA fragment was then purified by electrophoresis of the Hha I digest of the plasmid in low melting point agarose.
• the ⁇ -globin gene fragment DNA band was extracted fromthe agarose by melting the gel at 60 C followed by extraction with phenol.
• the thus obtained ⁇ - globin gene fragments contained approximately 50 micrograms of DNA per milliliter.
• the ⁇ -globin gene sequence contained a 223 base pair control sequence upstream from the cap site of the structural gene which included a TATA sequence (Goldberg, M., Ph.D. thesis, Stanford University (1979) and the CAAT sequences (Efstratiadis, A., et al, Cell, 21:653-668
• Injection pipets with an external diameter of about 1 micrometer and holding pipets with an external diameter of about 60-70 micrometers were prepared from pyrex tubing as described in Proc. Natl. Sci. U.S.A., 74:5657-5661 (1977). Manipulation of the pipets for holding and subsequence injection of the zygote was accomplished using Leitz micro- manipulators and paraffin oil filled Hamilton microsyringes. A small drop each of culture medium with 5-6 zygotes and of rabbit ⁇ -globin chromosomal gene solution were placed on a special microscope slide and covered with paraffin oil.
• ⁇ -globin gene solution which is approximately 20,000 DNA sequences
• a zygote was positioned onto the holding pipet so that the male pronucleus was in juxtaposition to the injection pipet for subsequent injection of the ⁇ -globin gene suspension into the pronucleus. After microinjection of all of the zygotes, they were removed from the drop of medium on the microscope slide and placed in cultu re tubes where they were allowed to develop for five days . The conditions of the preimpiantation development is described in Biol . Rep rod . , 8:420-426 (1973) .
• fiJREA ⁇ O PI Hemoglobin was prepared from blood samples taken from each of the mice developed from the rabbit ⁇ -globin gene microinjected F x hybrid mouse zygotes and analyzed by agarose immunod ⁇ ffusion against F 1 hybrid (C57BL/6JxLT/Sv) mouse antirabbit hemoglobin antiserum. The three samples which showed the most distinct positive reaction with the antiserum were pooled and used in the immunodiffusion analyses hereinafter described.
• the agarose gel immunodiffusion of Figure 3 shows identity between one of the dissociated rabbit globin chains and a molecular species present in the erythrocytes of mice developed from the ⁇ -globin gene microinjected zygotes.
• the hemoglobin of the beneficialally transformed mice is comprised apparently of at least one rabbit ⁇ -globin and mouse c-glob ⁇ ns.
• mice that had been genetically transformed with the rabbit ⁇ -globin gene also have an abnormally high percent, about 6 percent, of reticulocytes in their blood .
• the control mice of the same inbred species and age have a reticulocyte concentration of about 0.5 percent of their blood cells .
• the high percentage of reticulocytes is indicative of thalassemia, an anemia resulting from the presence of excess rabbit ⁇ - globin .
• mice that were genetically transformed by the rabbit ⁇ -globin gene have produced offspring which also phenotypically express the rabbit ⁇ - glob ⁇ n gene.
• Each hemoglobin preparation was prepared as a purified hemolysate with blood from New Zealand white rabbits , normal F x hybrid ( C57BL/6 LT/Sv) mice and F hybrid
• O PI mice developed from zygotes microinjected with rabbit ⁇ -globin gene DNA sequences.
• the erythrocytes for each hemoglobin • preparation were washed exhaustively with phosphate buffered saline, lysed in distilled water and centrifuged at 12,000 times gravity for 10 minutes to remove the erythrocyte membrane.
• the hemoglobin solutions Prior to application of the hemoglobin sample to agarose immunodiffusion plates, the hemoglobin solutions were filtered th rough a 0.45 micro filter and adjusted toa concentration of 100 mg/ml .
• the mouse antirabbit hemoglobin antiserum was raised in
• Fi hybrid mice (C57BL/6Jx LT/Sv) mice. Rabbit hemoglobin in distilled water was ⁇ emulsified with an equal volume of complete Freund's adjuvant and each animal received a 500 microgram priming dose, subcutaneously, divided among four sites. After one and two weeks all animals received second and third injections of 10 micrograms of rabbit hemoglobin emulsified in incomplete Freund's adjuvant administered subcutaneously and intraperitoneally, respectively. After four weeks each animal was injected intraperitoneally with 600 micrograms of rabbit hemoglobin in complete Freund's adjuvant. The immunized animals were bled after week five and the serum separated, tested against rabbit hemoglobin and preserved in the presence of sodium azide.
• Free rabbit globin chains were prepared from a rabbit hemolysate by acetone extraction of the heme iron complex resulting in dissociation of the a and ⁇ protein subunits . Five hundred micrograms of packed, washed, rabbit erythrocytes were lysed in 2 mill ⁇ l ⁇ ters of distilled water and the resulting hemolysate dripped slowly into a stirred solution of acetone and 1 .2 milliliters of concentrated HC1 at -70 C .
• the resulting precipitated rabbit globin chains were allowed to stand for 15 minutes at -70 C in the extraction mixtu re after which they were washed twice with -20 C acetone and dried under vacuum at liquid nitrogen temperatures .
• the dried precipitate was dissolved in 2 milliliters of distilled H 2 0 and dialysed exhaustively against phosphate buffered saline. Rabbit hemoglobin, 30 milligrams, was coupled to
• Sepharose 4B Gel beads by reaction with 1 gram washed CNBr-activated Sepharose 4B from Pharmacia Fine Chemicals, I nc. in 0. 1M NaHCO 3 and 0.5M NaCl at a pH of 8.0, for 3 hours at room temperature. Following the coupling reaction, the remaining active groups on the gel beads were reacted with 1M Tris, pH 8.0, and the hemoglobin Sepharose gel was washed exhaustively to remove any unbound hemoglobin . The gel-immobilized rabbit hemoglobin was used to prepare a miniature column for the selective removal of antirabbit hemoglobin antibodies from immunized mouse serum.
• Reactivity of transformed mouse hemoglobin antigens with mouse antirabbit hemoglobin antiserum was detected by double diffusion analysis on 1 percent agarose slides.
• Antigen wells contained 15 microl ⁇ ters of each hemoglovin antigen (100 mg/ml) and the antibody well contained 15 microliters of antirabbit hemoglobin antiserum. Each well was refilled twice during the 24 hour incubation at 37 C. Reactivity was characterized by the production of a band of precipitation between the hemoglobin of the transformed mice and the mouse antirabbit hemoglobin antiserum.

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• 1982
  • 1982-06-08 WO PCT/US1982/000780 patent/WO1982004443A1/en not_active Application Discontinuation
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