WO2016163386A1 - 異個体由来の配偶子を生産する非ヒト大型哺乳動物又は魚類の作出方法 - Google Patents
異個体由来の配偶子を生産する非ヒト大型哺乳動物又は魚類の作出方法 Download PDFInfo
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- WO2016163386A1 WO2016163386A1 PCT/JP2016/061224 JP2016061224W WO2016163386A1 WO 2016163386 A1 WO2016163386 A1 WO 2016163386A1 JP 2016061224 W JP2016061224 W JP 2016061224W WO 2016163386 A1 WO2016163386 A1 WO 2016163386A1
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Definitions
- the present invention relates to a method for producing a non-human large mammal or fish that produces gametes derived from different individuals.
- KO mice are an important model organism.
- ES cells embryonic stem cells
- SCNT somatic cell nuclear transfer
- An object of the present invention is to provide a novel means that enables stable mass production of knockout individuals even in large animals.
- Non-Patent Documents 6 to 9, Patent Documents 1 to 3 This technique is known as a blastocyst complementation technique.
- the nanos3 gene is a germ cell differentiation-related gene that is specifically expressed in primordial germ cells, but it has been reported that when nanos3 is knocked out in mice, germ cells (sperm / egg) are not formed ( Non-patent document 10).
- the inventors of the present application pay attention to these technologies, and when a chimeric embryo is produced using a nonos3-KO / SCNT embryo as a host embryo and a specific gene KO / SCNT embryo-derived cell as a donor cell, the sperm and egg of the individual born are We found that all the specific genes can be in the KO state, and that it is possible to stably obtain large KO animals of the specific gene by mating (artificial insemination and in vitro fertilization).
- the present invention is a method for producing a non-human large mammal or fish that produces gametes derived from different individuals, wherein the function of the nanos3 gene derived from the first non-human large mammal or fish is inhibited.
- a chimeric embryo is prepared by transplanting at least one pluripotent cell derived from a second non-human large mammal or fish into an embryo at the cleavage stage having the same genome, and the chimeric embryo is generated to produce an individual.
- Providing a method comprising: obtaining.
- the present invention also provides a method for producing an egg of a non-human large mammal or fish, which comprises recovering the egg from a female individual of the non-human large mammal or fish produced by the production method of the present invention. .
- the present invention provides a method for producing sperm of a non-human large mammal or fish, which comprises collecting sperm from a male individual of the non-human large mammal or fish produced by the production method of the present invention. . Furthermore, this invention provides the production method of the fertilized egg of a non-human large mammal or fish including fertilizing the egg and sperm produced by the production method of the said invention, and obtaining a fertilized egg. Furthermore, the present invention provides the production of a non-human large mammal or fish, which comprises obtaining a progeny of a male and female non-human large animal or fish produced by the production method of the present invention by natural mating, artificial insemination or in vitro insemination. Provide a method.
- the present invention it is possible to stably supply an individual in which a desired specific gene has been knocked out, even in a large animal in which stable mass production of a knockout individual has been extremely difficult.
- Stable mass production of knockout individuals is one aspect of the present invention, and according to the present invention, non-human animals having desired genetic characteristics other than knockout can be stably supplied at low cost.
- Specific applications of the present invention include the following.
- Nanos3-KO cell nuclear transfer embryos are injected with pluripotent cells (ES-like cells, blastomeres, etc.) derived from individuals in which the desired gene (A gene) has been KOed. Complement and generate individuals.
- a gene KO sperm is stably obtained from males, and A gene KO ovum is stably obtained from females.
- a gene KO individuals can be stably mass-produced.
- the SRY gene is a gene on the Y chromosome that serves to differentiate the gonad primordia into testis. If the SRY gene is inactivated, females will be born even if Y sperm is fertilized. When the birth of a female is desired in the prior art, it is necessary to select spermatozoa with a flow cytometer, but the sperm vitality is reduced by this selection operation.
- the target animal in the present invention is a non-human large mammal or fish.
- non-human large mammals and fish may be collectively referred to as “non-human animals”.
- the non-human large mammal that is the subject of the present invention can typically be a livestock animal.
- the term “large” is intended to exclude small mammals, and mammals that can be classified as medium-sized animals when classified in detail are also included in the non-human large mammals referred to in the present invention.
- Specific examples of non-human large mammals include various ungulates including artiopods such as cows, pigs, sheep, goats, wild boars, deer, camels, hippopotamus, and odd-hoofed animals such as horses, rhinoceros, tapirs, and the like
- laboratory animals include non-rodents except for rabbits such as monkeys and dogs, which are generally classified as large animals. Cattle can be mentioned as a particularly preferred example, but is not limited thereto.
- the target fish can typically be a cultured fish.
- aquaculture techniques have been developed for various food fish.
- Specific examples of fish to be used in the present invention include, but are not limited to, tuna, yellowtail, mackerel, skipjack, and horse mackerel.
- an embryo having a genome in which the function of the nanos3 gene is inhibited, and a pluripotent cell derived from another non-human animal individual is transplanted (injected) into an embryo at the cleavage stage derived from a non-human animal. )
- germ cells gametes, that is, sperm / egg
- a germ cell is complemented by a competent cell, and an animal individual producing a germ cell derived from the separate body can be obtained.
- the non-human animal non-human large mammal or fish on the side that inhibits the function of the nanos3 gene
- first non-human animal is referred to as “first non-human animal”, and is pluripotent for complementing germ cells.
- second non-human animal The non-human animal from which the cells are derived is referred to as “second non-human animal”.
- the first non-human animal and the second non-human animal may be animal individuals belonging to the same species or the same breed (for example, cows, Japanese black breeds, etc.), or animals of different or different breeds. Also good (eg sheep and cattle, Japanese black and Holstein).
- “inhibiting the function of a gene” means reducing or deleting the production or accumulation of the originally encoded mRNA or protein by modifying at least a part of the gene region on the genome. This includes everything from loss of gene function to complete loss of function. Genetic modification methods for inhibiting the function of a specific gene are widely known in this field, and those skilled in the art can appropriately select and execute them. Broadly classified, there are gene disruption methods (knock-out methods) for deleting gene functions and gene knock-down methods for reducing gene functions. Specific examples of knock-down methods include antisense methods and RNAi. .
- the inhibition of the function of the nanos3 gene in the present invention is preferably a functional defect caused by disruption (knockout) of the nanos3 gene.
- knockout For example, knocking out the nanos3 gene by deleting the coding region or promoter region of the nanos3 gene in both alleles of the genome, or introducing a mutation such as substitution or insertion so that normal nanos3 protein cannot be produced. Can do.
- all or part of the nanos3 gene coding region may be replaced with a marker gene sequence such as drug resistance or fluorescent protein.
- gene knockout As a specific method of gene knockout, there can be mentioned a knockout method by homologous recombination using a targeting vector described in the following Examples.
- Other gene knockout methods include zinc finger nuclease (ZFN) method (Porteus, MH et al. Gene targeting using zinc finger nucleases. Nat. Biotechnol. 23, 967-973 (2005).), TALEN method (Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757-761 (2010).) and CRISPR / Cas9 method (Sander, JD et al. CRISPR-Cas systems for editing, regulating and targeting genomes.
- ZFN zinc finger nuclease
- TALEN method Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757-761 (2010).
- CRISPR / Cas9 method ander, JD e
- An appropriate knockout method may be selected according to the type of non-human animal used for gene knockout.
- a method using a targeting vector requires a step of reconstructing an embryo by somatic cell nuclear transfer, as described later, but an animal with low somatic cell nuclear transfer efficiency, such as a pig, or an embryo reconstructing by somatic cell nuclear transfer.
- the ZFN method, TALEN method, and CRISPR / Cas9 method that do not require a somatic cell nuclear transfer step can be preferably used.
- the upstream and downstream genomic sequences of the region to be deleted are amplified from the genomic DNA of the target organism by PCR to prepare the upstream and downstream homologous regions.
- Homologous region and marker gene are sequentially inserted into an appropriate plasmid vector to construct a targeting vector comprising a DNA construct for gene disruption arranged in the order of upstream homologous region-marker gene-downstream homologous region, and electroporation it. It may be introduced into somatic cells derived from the target organism (fibroblasts, etc.) by a conventional method such as.
- a gene disruption construct is introduced into a desired position on the genome by homologous recombination, and a mutant allele in which part or all of the nanos3 gene is replaced with a marker gene is generated.
- the size of the upstream homologous region and the downstream homologous region affects the efficiency of homologous recombination, and a homologous region having a large size is used for a low-efficiency species.
- a commonly used homologous region is about several kb in size.
- One homologous region is generally about 1 to 3 kb (short arm) and the other homologous region is about 5 kb or more (long arm), but both may be about 5 kb in size.
- the nucleotide sequences shown in SEQ ID NOs: 3 and 4 are the genomic regions upstream and downstream of the bovine nanos3 gene. When knocking out the bovine nanos3 gene, these regions or their partial regions of appropriate size are homologous. It can be used as an area.
- the nanos3 gene has been cloned in various animals including invertebrates and vertebrates.
- the sequence information of the identified nanos3 gene such as humans and mice, is used.
- Genome sequence information required for functional inhibition of the nanos3 gene such as homologous regions to be incorporated into targeting vectors by searching the entire genome sequence information (shotgun sequence, etc.) and identifying the predicted nanos3 gene region Can be obtained.
- a positive selection marker that imparts drug resistance In mammalian cells, the frequency of introduction of gene disruption constructs by homologous recombination into the genome is much lower than the frequency of random introduction without homologous recombination. Therefore, in the knockout of the nanos3 gene in the present invention, it is preferable to use a positive selection marker that imparts drug resistance and a negative selection marker that imparts drug sensitivity in combination.
- a marker gene linked between two homologous regions is defined as a positive selection marker gene, outside the two homologous regions (5 ′ side of the upstream homologous region or 3 ′ side of the downstream homologous region) ) With a negative selection marker gene.
- the construct is introduced into the genome by homologous recombination, the region outside the homologous region of the construct is not introduced into the genome, so that drug sensitivity is not imparted by the negative selectable marker gene.
- a negative selectable marker gene is also introduced into the genome, so that drug sensitivity is imparted to such transformed cells. Therefore, if a construct for gene disruption is introduced into a somatic cell derived from the first non-human animal and then screened with a positive selection marker and a negative selection marker, the construct is introduced at an appropriate position by homologous recombination and the nanos3 gene is destroyed. The selected cells can be efficiently selected.
- marker genes include neomycin resistance gene, blasticidin resistance gene, puromycin resistance gene and the like as positive selection markers, and thymidine kinase gene and diphtheria toxin A fragment as negative selection markers.
- DT-A thymidine kinase gene and diphtheria toxin A fragment as negative selection markers.
- a knockdown process of the BML gene may be performed.
- knockdown treatment of the BML gene increases the efficiency of homologous recombination (So S et al. Genes to Cells 2006; 11 (4): 363-371.).
- knockdown of the BML gene is also effective in improving the homologous recombination efficiency in the non-human animals targeted in (1).
- sequence information of BML genes is also known, and nucleic acid reagents for knocking down BML genes of various animal species are commercially available. Those skilled in the art can appropriately use such commercially available products to knock BML genes. Down processing can be implemented.
- Somatic cell nuclear transfer technology has become an established technology even in large mammals (Nature, 385, p.810-813, 1997; Science, 282 (5396), p.2095-2098, 1998; Science, 298, p 1188-1190, 2000; Nature, 407, p.86-90, 2000; Nat Biotechnol., 18, P.1055-1059, 2000; Cloning Stem Cells 9, 571-580 (2007) and the like).
- a reconstructed embryo derived from the first non-human large mammal having a genome in which the function of the gene is inhibited can be obtained.
- the reconstructed embryo is activated and cultured until the cleavage stage, and at least one pluripotent cell derived from a second non-human large mammal in which the nanos3 gene is normal (function is not inhibited) Inject chimera embryos by injection.
- the number of pluripotent cells to be injected may be at least one. Usually, a plurality of cells, for example, about several to tens of cells are injected.
- the pluripotent cell derived from the second non-human animal is not particularly limited as long as it has differentiation pluripotency.
- ES cells and iPS cell lines can be used as long as ES cells and iPS cell lines are established, and fertilized egg blastomeres can be used in unestablished animal species, for example. .
- the stage of development of the first non-human animal-derived embryo when injecting pluripotent cells is not particularly limited as long as it is the cleavage stage, and it may be any stage from the 2-cell stage to the blastocyst stage.
- it can be at the 4-cell stage, 8-cell stage, 16-cell stage, morula stage, or blastocyst stage.
- the injection is preferably performed from the morula stage to the blastocyst stage.
- germ cells gametes, ie, eggs or sperm
- a non-human large mammal that produces such a gamete derived from a different individual can be obtained.
- the surrogate mother for transplanting the chimeric embryo is usually a female individual of the same species as the first non-human large mammal. For example, if the first non-human large mammal is a sheep and the second non-human large mammal is a cow, the surrogate mother for transplanting the chimeric embryo is usually a female sheep.
- the gene knockout methods ZFN, TALEN, and CRISPR / Cas9 which are gene knockout methods that can be used in addition to the targeting vector method, are all DNA recognition sites (ZFN method) designed to specifically recognize the desired base sequence.
- ZFN method DNA recognition sites
- TALEN method uses DNA binding domain of TAL effector derived from plant pathogen Xanthomonas
- CRISPR / Cas9 method uses artificial nuclease fused with nuclease containing guide RNA containing complementary sequence to DNA sequence to be cleaved) It is a technique.
- Non-Homologous End Joining NHEJ
- HDR homologous recombination repair
- an artificial nuclease designed to target the nanos3 gene is introduced into a fertilized egg derived from the first non-human animal to destroy the nanos3 gene, thereby producing a homozygous fertilized egg.
- a fertilized egg (embryo) having a genome in which the nanos3 gene is knocked out can be obtained, the somatic cell nuclear transfer operation need not be performed. Therefore, in the case of an animal species with low efficiency of somatic cell nuclear transfer, these methods can be preferably used rather than the gene knockout method using a targeting vector. These methods can also be preferably used in the production of fish nanos3 gene knockout bodies.
- a chimeric embryo may be prepared by transplanting pluripotent cells derived from a human animal. If this chimeric embryo is generated, a non-human animal individual producing gametes derived from different individuals can be obtained. In the case of mammals, a chimeric embryo may be transplanted into a surrogate mother (temporary parent) to obtain a litter. In the case of fish, of course, this embryo transfer step is unnecessary.
- Progenies of non-human animals that produce gametes derived from different individuals obtained by generating chimeric embryos can be obtained by natural mating of such male and female of non-human animals, or by artificial insemination or in vitro fertilization .
- in vitro fertilization a fertilized egg that has been in vitro fertilized is usually transplanted to a female individual of a non-human animal that produces a gamete derived from the different individual produced by the method of the present invention.
- the fertilized egg may be transplanted into an individual of the same species as the second non-human animal.
- livestock sperm or eggs having a high breeding cost can be produced into a livestock having a low breeding cost.
- sheep and goats are stronger than cattle and can withstand rough meals, so the management cost is low, and they are precocious and have good breeding efficiency. Therefore, if sheep and goats are used as the first non-human animal and cattle are used as the second non-human animal, it is possible to cause the sheep and goats to produce bovine gametes. It becomes possible to provide sperm and fertilized eggs for artificial insemination.
- horse mackerel or mackerel is used as the first non-human animal and tuna is used as the second non-human animal, the horse mackerel or mackerel can make tuna gametes. It is possible to mass-produce fry at low cost.
- a gamete having a desired genetic characteristic possessed by a second non-human animal different from the non-human animal can be produced.
- a non-human animal strain that produces such a gamete is established, male and female mating (natural mating, artificial insemination, or in vitro fertilization) can be used to produce non-human strains with the desired genetic characteristics regardless of somatic cell cloning techniques. Mass production of individual human animals is possible.
- the “desired genetic characteristics” include both genetic characteristics naturally occurring in the second non-human animal and artificial genetic modifications.
- the characteristic that a breeding value is very high for example, a body growth ability is very high
- An artificial genetic modification can be, for example, inhibition of the function of a desired gene, typically a knockout of the desired gene.
- a non-human animal gamete having a desired gene knocked out is produced in another non-human animal, the desired gene is knocked out in a second non-human animal-derived pluripotent cell used for the production of a chimeric embryo. It only has to be.
- a pluripotent cell derived from a second non-human animal having a genome in which a desired gene is knocked out is basically knocked out of the nanos3 gene using an appropriate cell derived from the second non-human animal. The same procedure can be used.
- a targeting vector a blastomere of a reconstructed embryo in which a desired gene is knocked out homozygously can be used as the pluripotent cell.
- a blastomere of a fertilized egg in which a desired gene is knocked out homozygously can be used as the pluripotent cell.
- an egg and sperm in which a desired gene is knocked out can be stably produced in a non-human animal individual in which the gene is not knocked out.
- a non-human animal in which a specific gene is knocked out can be produced by natural mating or artificial insemination or in vitro fertilization of a non-human animal producing a gamete in which a desired gene is knocked out produced by the method of the present invention. It can be obtained stably. According to the present invention, it is possible to stably supply specific gene knockout individuals even in large animals.
- somatic cell nuclear transfer cloning technology By using somatic cell nuclear transfer cloning technology and gene recombination technology, it has become possible to produce gene knockout (KO) individuals even in medium and large livestock. These techniques are the most effective techniques for functional analysis of specific genes required for gene breeding of livestock. However, the efficiency of individual production using somatic cell nuclear transfer clone technology is very low, and it is impossible to stably produce a large number of KO individuals at present. Recently, when a mixed embryo is created by mixing a gene KO early embryo and a normal embryo in a mouse, the mixed embryo complements the defects (cell differentiation disorder, organ defects, etc.) caused by KO embryo-derived cells and develops and born normally. Has been reported (Kobayashi et al., Cell 142, 787-799 (2010)).
- Bovine nanos3 gene genomic DNA KO vector construction The germ cell differentiation-related gene nanos3 gene KO vector was constructed based on the genomic structure information of bovine nanos3 gene published in NCBI database.
- a KO vector (pNOS3-KOn) used for hetero KO manipulation
- the genomic region surrounding the nanos3 gene (1.5 kb and 6.5 kb fragments: FIG. 2) was cloned by PCR cloning.
- template DNA genomic DNA extracted from Japanese black beef fetal fibroblasts was used.
- sense primer CTCTCCGTTGCATCCATGCC (SEQ ID NO: 6)
- antisense primer AGCCACTGACCTTCCAGCTGAC (SEQ ID NO: 7) )It was used.
- a sense primer: GGACAAGGTATCGTGAACTGC (SEQ ID NO: 8) and an antisense primer: AACACGAGGAGCACCTTCTTGC (SEQ ID NO: 9) are used.
- the pNOS3-KOn vector was constructed by inserting a PGK-neo unit (neomycin resistance gene having a PGK promoter) as a selection marker so as to delete the entire nanos3 gene (exons 1 and 2).
- the pNOS3-KOn vector has a short arm of 1.5 kS on the 5 ′ side of the PGK-neo unit and a long arm of 6.5 kL on the 3 ′ side, and a negative selection gene marker MC1-TK (5 ′ side of 1.5 kS).
- MC1-TK 5 ′ side of 1.5 kS.
- a herpes thymidine kinase gene with MC1 promoter was constructed ( Figure 2).
- KO-knock-in (KI) vector pNOS3-
- a vector used for homo-KO manipulation in which part of the region encoding the protein of the nanos3 gene is replaced with cDNA encoding the fluorescent protein kusabira orange (huKO) huKO-KIb) was constructed (Fig. 2).
- the cDNA fragment of wedge orange was synthesized by commissioning Eurofin Genomics and used for vector construction.
- the short arm sequence (2.0 k ⁇ bovine genome sequence + 0.66 k huKO + 0.35 k bovine genome sequence consisting of huKO cDNA) is shown in SEQ ID NO: 5.
- SEQ ID NO: 5 2049 to 2709 nt is the cDNA sequence of huKO.
- the selection marker used was a CAG-bsr unit (a blasticidin S resistance gene having a CAG promoter) (FIG. 2).
- knockdown treatment of bovine BML gene was also performed.
- the knockdown treatment of the bovine BML gene was performed using stealth RNA for bovine BML (synthesis position 2656) produced by consigning to Invitrogen according to the method of the previous report (Chiyo, Zhuchu Lab report, 1501-604 (2009)). It was.
- the pNOS3-huKO-KIb vector was introduced into the nanos3 hetero KO fetus-derived cell line (# 3933 strain) and the medium containing two sorts of drugs (neomycin: G418 and blasticidin S) was used. Culture and establishment were performed.
- PCR analysis for KO determination was performed according to a conventional method.
- the base sequence of the primer used for the analysis is shown below.
- P1 AACACGGTGAAGCTCACTTAGG (SEQ ID NO: 10)
- P2 CATGCTCCAGACTGCCTTGG (SEQ ID NO: 11)
- P3 CTCTCCGTTGCATCCATGCC (SEQ ID NO: 12)
- P4 CTTCATCTCGGGCTTGATCGTCG (SEQ ID NO: 13)
- P5: GCTTCATCCTTGAGCACGTGG SEQ ID NO: 14
- P6 CCACGTGCTCAAGGATGAAGC (SEQ ID NO: 15)
- P7 CTGATACGTAAGCCTAGCTACTCG (SEQ ID NO: 16)
- the set region of each primer is as shown in FIG. 4 and FIG.
- P3-P2 amplifies a part of the construct in the hetero-KO vector
- P1-P2 detectts an allele in which the hetero-KO construct is inserted at the correct position
- P3-P4 amplifies part of the construct in the homo KO vector
- P1-P4 the homo KO construct is inserted in the correct position
- Allele was detected
- P1-P5 wt allele was detected
- P6-P7 wt allele was detected
- Somatic cell nuclear transfer and fetal recovery A cell line in which hetero- or homo-KO of the nanos3 gene was confirmed by PCR analysis was used as a nuclear donor, and a previous report (Ideta, A. et al. Cloning Stem Cells 9, 571-580 ( 2007)), somatic cell nuclear transfer operation was performed to prepare a nuclear transfer embryo (reconstructed embryo). The procedure is briefly described as follows.
- Follicular ova were aspirated from bovine ovaries derived from slaughterhouses and matured for about 20 hours. After detaching cumulus cells with hyaluronidase (Sigma), ova whose release from the first polar body was confirmed were selected. Insert a nuclear donor into the periplasmic space of the enucleated recipient egg, fuse the cells by electrical stimulation, and perform artificial activation treatment with calcium ionophore (Sigma) to promote the development of reconstructed embryos. did. Thereafter, in vitro culture was performed in the development medium and the development was observed.
- Nuclear transfer embryos were transplanted into recipient cows, about 200-day-old fetuses were removed by caesarean section, and the ovaries of the fetuses were observed. After fixing the ovarian tissue with 10% neutral buffered formalin solution, tissue sections were prepared by a paraffin embedding method. Hematoxylin-eosin (HE) staining was performed, and the tissue was observed using an optical microscope.
- HE Hematoxylin-eosin
- Nanos3-KO nuclear transfer embryos (morula stage embryos) were injected with Holstein in vitro fertilized embryo blastomeres (7-10) and cultured in vitro for 2 days. After transplanting the grown chimeric embryo into a recipient cow, about 140-day-old fetuses were removed by caesarean section and the ovaries of the fetuses were observed. Moreover, the chimera ratio (Holstein cell content ratio) of each organ of the chimeric fetus was investigated by a real-time PCR method.
- the bovine nanos3 gene was estimated from genomic information (XM_002688743, SEQ ID NO: 1), but when the bovine exon was estimated by comparing the sequence information shown in XM_002688743 with the exon information of the human nanos3 gene, two exons were found. Estimated. As a result of analyzing the sequence of these estimated exons (1 and 2) by NCBI BLAST, the chromosome (No. 7) in which each exon of bovine nanos3 is located and the surrounding gene sequence (NC007305.5, region 10061880) was gotten. From the above results, the presence of the nanos3 gene could be confirmed in cattle, and the gene structure was found to be composed of two exons as in the mouse (FIG. 2). Cloning of the genomic region around the nanos3 gene (1.5 kb and 6.5 kb fragments: FIG. 2) for the construction of the nanos3 gene KO vector was performed by PCR cloning.
- the KO vector was constructed using a positive-negative selection type and deleting the entire nanos3 gene (FIG. 2).
- PGK-neo was used as a drug resistance gene for positive selection.
- MC1-TK was used as a drug susceptibility gene for negative selection (FIG. 2: pNOS-KOn).
- a KO-huKO-KI vector was constructed in which a part of the region encoding the protein of the nanos3 gene was replaced with a cDNA encoding the fluorescent protein Xavira orange (huKO).
- CAG-bsr was used as a drug resistance gene for selection (FIG.
- the # 4-68 strain which had good cell growth, was used to produce, collect, and establish cell lines of nuclear transfer clone fetuses. As a result, one fetus was successfully created and collected, and a fetus-derived cell line was successfully established (# 3933 strain), and it was confirmed that it was hetero KO in genomic PCR analysis (FIG. 4).
- a KO-huKO-KI vector (pNOS3-huKO-KIb) was introduced into the established hetero KO cells (# 3933 strain) to try to establish a homo KO cell line.
- 15 well was determined to be homo-KO-huKO-KI among the 221 well investigated (Table 2).
- 15 well of 15 well determined to be homo-KO-huKO-KI it is a cell that is derived from a single colony, has good growth, and undergoes homologous recombination reaction at the correct position. This was suggested by PCR analysis (# 2-36 strain) (FIG. 5).
- FIG. 6 shows a histological image of the ovary of nanos3 homo KO fetus.
- Fig. 6d In non-KO almost equal-aged fetuses, numerous primary follicles are found in the ovarian cortex and germ cells are normally formed (Fig. 6d), but follicular cells can be confirmed in the ovaries of nanos3 homo-KO fetuses. None ( Figure 6a-c). From this, it was clarified that the primordial germ cells of nanos3 knockout cattle were killed by apoptosis in the early embryonic period, and the living body (194-day-old fetus) was completely deficient in germ cells.
- FIG. 7 shows a histological image of the ovary (141 days old). In one of the two chimeric fetuses, a primary follicle could be confirmed in the ovary. From this, it became clear that the germ cells derived from donor cells were complemented in the ovaries of nanos3-KO cattle by the blastocyst complementation method.
- the chimera ratio (Holstein cell content rate) of each organ of the chimeric fetus was examined by real-time PCR. As a result, the brain was 12.1%, the heart was 20.2%, the liver was 1.8%, the uterus was 22.4%, and the ovary was 15.8%.
- the fetal bovine produced here is a chimera of Japanese black and Holstein, and Holstein germ cells are formed in the ovary.
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Abstract
Description
nanos3-KO細胞核移植胚に所望の遺伝子(A遺伝子)がKOされた個体由来の多能性細胞(ES様細胞、割球など)を注入して生殖細胞を補完し、個体を発生させる。雄からはA遺伝子KO***が、雌からはA遺伝子KO卵子が安定的に得られる。両者の交配(自然交配、人工授精、又は体外受精)により、A遺伝子KO個体を安定的に大量生産できる。
nanos3-KO細胞核移植胚に望ましい遺伝的特性を有する個体由来の多能性細胞(ES様細胞、割球など)を注入して生殖細胞を補完し、個体を発生させる。該個体は、望ましい遺伝的特性を有する***又は卵子を作る。***数が多い系統で該個体を作れば(つまり、nanos3-KO細胞株及び核移植レシピエントとしてそのような系統を用いればよい)、望ましい遺伝的特性を有する***、卵子及び受精卵を安価に大量生産することができる。
ヤギやヒツジのnanos3-KO/核移植胚に優れた増体能を有する黒毛和牛由来の多能性細胞を注入して生殖細胞を補完し、個体(ヤギ、ヒツジ)を発生させる。該個体(ヤギ、ヒツジ)は、優れた増体能を有する黒毛和牛の***又は卵子を作る。管理コストが安価なヤギやヒツジから黒毛和牛の受精卵を大量に製造できる。哺乳動物のみならず、海洋生物でも可能である。例えば、アジやサバでnanos3-KO/核移植胚を作ってマグロの生殖細胞を補完し、マグロの***・卵子を作るアジやサバを得ることができる。このアジ・サバを飼育すればマグロが得られる。
SRY遺伝子は生殖腺原基を精巣に分化させる働きをするY染色体上の遺伝子である。SRY遺伝子を不活化すると、Y***が受精しても雌が生まれるようになる。従来技術で雌の誕生を希望する場合、フローサイトメーターによる***の選別が必要になるが、この選別操作により***の活力は低下してしまう。本発明の方法により、SRYをノックアウトした核移植胚をドナーとしてnanos3-KO胚の生殖細胞を補完することで、Y***が不活化した雌しか生まれない大型動物系統を作出することができる。
体細胞核移植クローン技術と遺伝子組換え技術を活用することにより、中大型家畜においても遺伝子ノックアウト(KO)個体の作出が可能となった。これらの技術は、家畜の遺伝子育種に必要とされる特定遺伝子の機能解析において最も有効な技術である。しかしながら、体細胞核移植クローン技術を用いた個体作出効率は非常に低い状況にあり、安定的に多数のKO個体を作出することは現状では不可能である。最近、マウスにおいて、遺伝子KO初期胚と正常胚を混合し混合胚を作製すると混合胚はKO胚由来の細胞によって生じる欠損(細胞分化障害および臓器欠損等)を補完し正常に発生・誕生することが報告された(Kobayashi et al., Cell 142, 787-799 (2010))。また、マウスにおいて生殖細胞分化関連遺伝子nanos3をKOすると卵子および***が形成されないことが報告されている(Tsuda et al., SCIENCE 301, 1239-1241 (2003))。本試験においては、先に示した胚盤胞補完法を利用し、nanos3遺伝子をKOした胚と特定遺伝子をKOした胚を混合し補完胚を作製した時、遺伝子KO卵子(または***)のみを形成する個体が作出できるかを検討する。当該技術が実証された場合、交配や人工授精により安定的にKO個体のみを作出することが可能となる(図1:技術の概略)。
ア.ウシnanos3遺伝子ゲノムDNA KOベクター構築
生殖細胞分化関連遺伝子nanos3遺伝子KOベクターの構築は、NCBIデーターベースに公開されているウシnanos3遺伝子ゲノム構造情報をもとに実施した。
ウシ(黒毛和牛)胎仔由来線維芽細胞(#906♀株)へのpNOS3-KOnベクターの導入・選別培養およびKO細胞株樹立は、既報(千代、飼中研報告、1501-622(2009)、及びSendai, Y. et al., Transplantation 81, 706-766 (2006))の方法に従い行った。また、ヒト細胞を用いた研究でBLM遺伝子のノックダウン処理が相同組換え効率を上げるという報告があることから(So S et al. Genes to Cells 2006; 11(4):363-371.)、nanos3ノックダウンにおける相同組換えの効率を上げる目的で、ウシBML遺伝子のノックダウン処理も行なった。ウシBML遺伝子のノックダウン処理は、既報(千代、飼中研報告、1501-604(2009))の方法に従い、インビトロジェン社に委託して作製したウシBML用stealth RNA(合成位置2656)を用いて行なった。ホモKO細胞株樹立においては、nanos3ヘテロKO胎仔由来細胞株(#3933株)にpNOS3-huKO-KIbベクターを導入し、2種の選別薬剤(ネオマイシン:G418およびブラストサイジンS)を含む培地で培養し樹立操作を行った。
P1: AACACGGTGAAGCTCACTTAGG(配列番号10)
P2: CATGCTCCAGACTGCCTTGG(配列番号11)
P3: CTCTCCGTTGCATCCATGCC(配列番号12)
P4: CTTCATCTCGGGCTTGATCGTCG(配列番号13)
P5: GCTTCATCCTTGAGCACGTGG(配列番号14)
P6: CCACGTGCTCAAGGATGAAGC(配列番号15)
P7: CTGATACGTAAGCCTAGCTACTCG(配列番号16)
PCR解析によりnanos3遺伝子のヘテロKO又はホモKOが確認された細胞株を核ドナーとして使用し、既報(Ideta, A. et al. Cloning Stem Cells 9, 571-580 (2007))に従い体細胞核移植操作を行ない、核移植胚(再構築胚)を調製した。手順を簡単に記載すると以下の通りである。
nanos3-KO核移植胚(桑実期胚)にホルスタイン種体外受精胚の割球(7~10個)を注入し、2日間体外培養した。成長したキメラ胚をレシピエント牛に移植後、約140日齢胎仔を帝王切開により取り出し、胎仔の卵巣を観察した。また、キメラ胎仔の各臓器のキメラ率(ホルスタイン細胞含有率)をリアルタイムPCR法で調査した。
(1)ウシnanos3遺伝子ゲノムのクローニングと遺伝子KOベクターの構築
マウスを用いたKO個体作出実験において、生殖細胞分化関連遺伝子nanos3をKOすると発生の初期段階に存在する始原生殖細胞の***能が低下し且つ生殖***への移動が起こらず、生まれたKO個体の卵巣および精巣内に卵子や***が形成されないことが報告されている(Tsuda et al., SCIENCE 301, 1239-1241 (2003))。また、最近の研究において、特定遺伝子をKOして臓器等が形成できない状態にした初期胚に正常初期胚に由来する未分化細胞を混合すると、発生中の混合胚において正常胚に由来する細胞がKO欠損細胞を補完し(胚盤胞補完)正常な臓器が形成されることがマウスおよびブタにおいて示された(Kobayashi et al., Cell 142, 787-799 (2010)、及びMatsunari et al., PNAS 110(12),4557-4562 (2013))。これらの結果は、nanos3遺伝子をKOした胚と特定遺伝子をKOした胚を混合し混合補完胚を作製した場合、特定遺伝子KO卵子(または***)のみを形成する個体が作出できる可能性を示しており、特定遺伝子KO個体の安定的生産系が実現する可能性も示唆している(図1)。本試験においては、この仮説を確認するため、ウシnanos3遺伝子KO個体作出に向けKO細胞株の樹立を試みた。
構築したKOベクター(pNOS3-KOn)を使用し、雌黒毛和牛胎仔由来線維芽細胞(#906株(♀))を用いてKO操作を実施し、まずヘテロKO細胞株を樹立した。KO株樹立試験を4回実施した結果、調査した411 well中、9 wellがKOと判定された(表1)。このKOと判定された9 wellのうち4 wellにおいては、単一のコロニーに由来し、正しい位置で相同組換え反応が起きている細胞が増殖していることが、詳細なPCR解析により示唆された(#2-4株、#4-24株、#4-25株、#4-68株)(図4)。これら4株のうち、細胞増殖性が良かった#4-68株を用いて核移植クローン胎仔の作出および回収・細胞株樹立を行った。その結果、1胎仔の作出・回収に成功し胎仔由来細胞株の樹立(#3933株)にも成功し、ゲノムPCR解析においてもヘテロKOであることが確認できた(図4)。
nanos3ホモKO胎仔の卵巣の組織像を図6に示す。非KOのほぼ同齢の胎仔では、卵巣皮質内に多数の一次卵胞が認められ、生殖細胞が正常に形成されるが(図6d)、nanos3ホモKO胎仔の卵巣内には卵胞細胞が確認できなかった(図6a-c)。このことから、nanos3ノックアウトウシの始原生殖細胞は胎生初期でアポトーシスにより死滅し、生体(194日齢胎仔)は完全に生殖細胞を欠損することが明らかとなった。
nanos3-KO核移植胚(桑実期胚)にホルスタイン種体外受精胚の割球(7~10個)を注入して作製したキメラ胚由来の胎仔(141日齢)の卵巣の組織像を図7に示す。2頭のキメラ胎仔のうちの1頭において、卵巣内に一次卵胞が確認できた。このことから、胚盤胞補完法によってnanos3-KOウシの卵巣内にドナー細胞由来の生殖細胞が補完されることが明らかとなった。
Claims (13)
- 異個体由来の配偶子を生産する非ヒト大型哺乳動物又は魚類の作出方法であって、第1の非ヒト大型哺乳動物又は魚類に由来する、nanos3遺伝子の機能が阻害されたゲノムを有する卵割期の胚に、第2の非ヒト大型哺乳動物又は魚類に由来する少なくとも1個の多能性細胞を移植してキメラ胚を調製し、該キメラ胚を発生させて個体を得ることを含む、方法。
- nanos3遺伝子の機能の阻害が、相同組換えによるnanos3遺伝子のノックアウトにより行われる、請求項1記載の方法。
- 前記卵割期が2細胞期~胚盤胞期である、請求項1又は2記載の方法。
- 前記多能性細胞は、所望の遺伝的特性を有する第2の非ヒト大型哺乳動物又は魚類に由来する細胞であり、所望の遺伝的特性を有する異個体由来の配偶子を生産する非ヒト大型哺乳動物又は魚類を作出する、請求項1ないし3のいずれか1項に記載の方法。
- 所望の遺伝的特性は、人為的な遺伝的改変である、請求項4記載の方法。
- 人為的な遺伝的改変は、所望の遺伝子のノックアウトである、請求項5記載の方法。
- 前記多能性細胞は割球である、請求項1ないし6のいずれか1項に記載の方法。
- 前記方法は、異個体由来の配偶子を生産する非ヒト大型哺乳動物の作出方法であり、前記キメラ胚を非ヒト代理母に移植して産仔を得ることをさらに含む、請求項1ないし7のいずれか1項に記載の方法。
- 前記第1の非ヒト大型哺乳動物及び前記第2の非ヒト大型哺乳動物の少なくとも一方がウシである、請求項8記載の方法。
- 請求項1ないし9のいずれか1項に記載の方法により作出された非ヒト大型哺乳動物又は魚類の雌個体から卵子を回収することを含む、非ヒト大型哺乳動物又は魚類の卵子の生産方法。
- 請求項1ないし9のいずれか1項に記載の方法により作出された非ヒト大型哺乳動物又は魚類の雄個体から***を回収することを含む、非ヒト大型哺乳動物又は魚類の***の生産方法。
- 請求項10記載の方法により生産された卵子と、請求項11記載の方法により生産された***を受精して受精卵を得ることを含む、非ヒト大型哺乳動物又は魚類の受精卵の生産方法。
- 請求項1ないし9のいずれか1項に記載の方法により作出された雌雄の非ヒト大型動物又は魚類の後代を自然交配、人工授精又は体外授精により得ることを含む、非ヒト大型哺乳動物又は魚類の生産方法。
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