WO2015199225A1

WO2015199225A1 - Genetic modification method for poultry primordial germ cells, genetically-modified poultry primordial germ cells, method for producing genetically-modified poultry, and poultry eggs

Info

Publication number: WO2015199225A1
Application number: PCT/JP2015/068523
Authority: WO
Inventors: 勲大石; 京子吉井; 貴寛田上; 裕鏡味; 大地宮原
Original assignee: 国立研究開発法人産業技術総合研究所; 国立研究開発法人農業・食品産業技術総合研究機構
Priority date: 2014-06-27
Filing date: 2015-06-26
Publication date: 2015-12-30
Also published as: JP6644276B2; JPWO2015199225A1

Abstract

The present invention provides a genetic modification method for poultry primordial germ cells, characterized by modifying a gene of the poultry primordial germ cell by genome editing.

Description

Genetic modification method of poultry primordial germ cells, genetically modified poultry primordial germ cells, production method of genetically modified poultry, and poultry eggs

The present invention relates to a genetic modification method for poultry primordial germ cells, a genetically modified poultry primordial germ cell, a method for producing genetically modified poultry, and poultry eggs.

Unlike other organisms, birds have huge egg yolks, so it is extremely difficult to perform microscopic manipulation on embryos, and poultry genetic manipulation using early embryos that have undergone several cell divisions immediately after fertilization is exceptional. It has hardly been done except for research. In addition, there is no report on a clone individual production technique. For this reason, it is difficult to realize the genetic manipulation technique used in other livestock and animals for poultry. On the other hand, in poultry, attempts have been made to genetically modify primordial germ cells that will differentiate into sperm and eggs in the future. Specially, only poultry has succeeded in establishing primordial germ cells that retain gametogenic potential, which has not been achieved in various mammals including humans, mice, and livestock. On the other hand, poultry primordial germ cell lines are extremely difficult to introduce and express foreign genes, or cannot be established if the serum used in the medium is different. Even if it can be established, it has many properties that are greatly different from other cultured cell lines, such as being unable to maintain culture for a long period of time (Non-Patent Documents 1-3). For this reason, it is difficult to apply the knowledge and techniques obtained from cultured cell lines of other organisms to poultry primordial germ cell lines, and a method suitable for primordial germ cell lines must be developed. In fact, only a few reports of primordial germ cell culture and genetic manipulation have been made since the first poultry primordial germ cell genetic modification. In addition, genome editing techniques and the like that have been made in early embryos and cultured cells in recent years have not been applied to poultry primordial germ cell lines. Genome editing is desired to be applied to poultry in order to enable gene knockout that does not leave foreign genes in the chromosome and knock-in that inserts foreign genes into the target site, but technical reports that enable this have been made. Absent. In particular, in order to produce genetically modified poultry individuals, high-efficiency gene knockout and knock-in are required, but even in general mammalian cultured cells, the efficiency of gene modification by genome editing varies widely, and even mammals It is unpredictable how efficiency should be achieved in a cultured primordial germ cell line that is significantly different in nature from the cultured cells.

Furthermore, while male primordial germ cell lines of poultry can be cultured for a long time, establishment and culture of female primordial germ cell lines have been extremely difficult. For example, Non-Patent Document 2 fails to establish a female primordial germ cell line, and Non-Patent Document 3 reports that the maintenance of a female primordial germ cell line becomes impossible after about 100 days of culture. For this reason, no genetic manipulation using a female primordial germ cell line has been reported so far.

Although many problems and issues to be solved remain, it has become possible to genetically modify poultry by using primordial germ cell lines. There is a big efficiency problem in manufacturing. The creation of genetically modified individuals using the current primordial germ cell line is achieved by genetically modifying male primordial germ cells, transplanting them into recipient embryos, then hatching them, and raising male chimeric individuals (G0) for about half a year. After maturation, this is mated with a wild-type female to obtain a heterozygous recombinant individual in this progeny (F1). In order to obtain a homozygote, it was necessary to sexually mature heterozygous male and female F1 individuals and to breed heterozygous progeny progenies, which required a great deal of time and labor.

As described in Non-Patent Document 4, for chicken gene knockout, there is only one report by homologous recombination using male primordial germ cells. However, since the method involves inserting a foreign gene by homologous recombination, When gene knockout is carried out using, chicken itself and chicken eggs become recombinant products, so there are high barriers for use as food, and gene knockout techniques by methods other than homologous recombination are required.

In addition, it is technically possible to knock in a foreign gene to a target locus using the homologous recombination technique described in Non-Patent Document 4, but the method of Non-Patent Document 4 provides 28% homologous recombination. The number of clones is limited to about 10 clones per 10 ⁷ cells, the possibility of not only homologous recombination but also random gene transfer is undeniable, and reproduction It is noted that the ability to differentiate into lineage is remarkably poor, and on average, only one out of 900 progeny homologous recombination individuals can be obtained. If a technique for overcoming these drawbacks and knocking in a foreign gene to a targeted locus more efficiently is developed, a gene knock-in chicken can be established more easily. For example, if the recombination introduction efficiency can be improved by 3 times or more (28% is about 84% or more), the efficiency of obtaining the progeny of recombination will be improved by 3 times or more, and it is expected that more than 1 recombination individual will be obtained for 300 progeny However, if the clone establishment efficiency is improved by a factor of about 100, it is considered that about 1 clone of knock-in primordial germ cells can be obtained per 10 ⁵ cells.

The main purpose of the present invention is to provide efficient poultry genetic modification technology.

The present invention provides the following methods for genetic modification of poultry primordial germ cells, methods for producing genetically modified poultry, methods for subculturing female primordial germ cells, and knock-in poultry eggs.
Item 1. A method for gene modification of poultry primordial germ cells, which comprises modifying genes of poultry primordial germ cells by genome editing.
Item 2. Item 2. The genetic modification method according to Item 1, wherein the poultry primordial germ cells are female primordial germ cells.
Item 3. Item 3. The gene modification method according to

Item

1 or 2, wherein the gene is an egg protein gene.
Item 4. Item 4. The gene modification method according to any one of Items 1 to 3, wherein the gene is modified by genome editing using TALEN or CRISPR.
Item 5. Item 5. The gene modification method according to Item 4, wherein the gene is modified by genome editing using CRISPR.
Item 6. Item 6. The gene modification method according to any one of Items 1 to 5, wherein the gene modification is gene knock-in, knock-out or partial deletion.
Item 7. Any one of Items 1 to 6, wherein the gene is modified by knock-in, a drug resistance gene is incorporated into the poultry primordial germ cell, and the primordial germ cell genetically modified based on the drug resistance gene is selected. The gene modification method according to Item.
Item 8. Item 1- characterized in that the gene is modified by knockout or partial deletion, a drug resistance gene is introduced into the poultry primordial germ cell, and the primordial germ cell genetically modified based on the drug resistance gene is selected. 7. The gene modification method according to any one of 6 above.
Item 9. Item 9. The gene modification method according to Item 7 or 8, wherein the drug resistance gene is a puromycin resistance gene (Puro ^r ) or a zeocin resistance gene (Zeo ^r ).
Item 10. Item 10. The gene modification method according to any one of Items 1 to 9, wherein genome editing is performed using a plasmid vector or a virus vector.
Item 11. Item 11. The gene modification method according to Item 10, wherein a plasmid vector or a virus vector is introduced into an early poultry embryo, the genome of the endogenous primordial germ cell is edited, and the endogenous poultry primordial germ cell is genetically modified.
Item 12. Item 11. A poultry primordial germ cell obtained by the genetic modification method according to any one of Items 1 to 10 and genetically modified by genome editing.
Item 13. Item 13. The poultry primordial germ cell of Item 12, which is a female primordial germ cell.
Item 14. A step of obtaining a genetically modified chimera individual by transplanting a genetically modified poultry primordial germ cell obtained by the method of any one of Items 1 to 10 to a blastoderm, blood or gonad region of an early poultry embryo, A method for producing genetically modified poultry, comprising the step of mating an individual with a wild type individual, a genetically modified individual or a genetically modified chimeric individual.
Item 15. A genetic modification comprising the step of maturing a chimeric individual comprising an endogenous poultry primordial germ cell genetically modified by the method according to Item 11 and mating with a wild type individual, a genetically modified individual, or another genetically modified chimeric individual Poultry production method.
Item 16. Item 16. The method according to Item 14 or 15, wherein the genetically modified poultry genotype is a mutant homo (-/-).
Item 17. Item 17. The method according to any one of Items 14 to 16, wherein the genetic modification is a knockout of at least one in ovo protein gene selected from the group consisting of ovalbumin, ovomucoid, ovomucin, ovotransferrin, and ovoinhibitor.
Item 18. Item 17. The method according to any one of Items 14 to 16, wherein the genetic modification is a heterogeneous or homozygous knock-in of a foreign gene in an egg protein gene, and the female genetically modified poultry egg contains an expression product of the foreign gene.
Item 19. Item 18. At least one in-vitro allergen protein selected from the group consisting of ovalbumin, ovomucoid, ovomucin, ovotransferrin, ovoinhibitor, ovoglobulin, and lysozyme, obtained from female genetically modified poultry produced by the method according to Item 17. Knockout poultry eggs that have been reduced or eliminated.
Item 20. Item 19. A knock-in poultry egg containing an expression product of a foreign gene obtained from a female genetically modified poultry produced by the method according to Item 18.
Item 21. Item 21. The knock-in poultry egg according to Item 20, wherein the foreign gene is a gene encoding a human-derived protein.
Item 22. Item 22. The knockin according to Item 20 or 21, wherein the foreign gene is selected from the group consisting of an antibody or fragment thereof, enzyme, hormone, growth factor, cytokine, interferon, collagen, extracellular matrix molecule, vaccine, agonistic protein, and antagonistic protein. Poultry eggs.
Item 23. A method for subculturing female primordial germ cells, wherein the medium is exchanged at normal pressure or under low gravitational acceleration.

According to the present invention, genetic modification of poultry primordial germ cells can be performed efficiently by genome editing. Puro ^r for knockout mutation introduced into the target sequence with greater than 90% efficiency by the like Zeo ^r are possible, one system in 1 ~ 5x10 ⁵ cells with a recombinant transfer efficiency 80-90% higher for the knock Clones can be established with the above efficiency. In the case of allergens such as ovomucoid and ovalbumin, the gene can be knocked out to produce eggs that do not contain allergens. In addition, an egg containing a large amount of a useful gene product can be produced by knocking in a foreign gene that is useful for a protein gene in the egg such as an ovomucoid or ovalbumin gene.

Poultry eggs obtained by applying genome editing technology to in ovo protein genes of poultry primordial germ cells remove genes with high allergenicity from egg white by making genes such as ovomucoid and ovalbumin homozygous. be able to. Moreover, a poultry egg having an expression product of a foreign gene in the egg can be obtained by knocking in the foreign gene under the control of the fallopian tube promoter.

The genetically modified poultry primordial germ cells obtained by the present invention are hatched after transplantation into a recipient embryo to obtain a chimeric individual (G0), and the gene is modified according to conventional methods such as mating if necessary Finished poultry can be obtained. Primordial germ cells that have been genetically modified by genome editing not only have high genetic modification efficiency, but also when a chimeric individual is obtained after transplantation into a recipient embryo and crossed to obtain a progeny, the progeny is 1. A genetically modified individual can be obtained with a high probability of 1 in 7 to 2.3 birds (knock-out and partial defect) and 1 in 3.5 to 3.8 birds (knock-in). A chimeric individual is not a progeny, and the next generation of the chimeric individual is a “progeny”. Among the progenies are genetically modified individuals (recombinant individuals) derived from transplanted primordial germ cells (donors), wild-type individuals (non-recombinant individuals) derived from transplanted primordial germ cells (donor), and wild derived from endogenous primordial germ cells Although there are type individuals, the present invention is characterized by a very high proportion of genetically modified individuals. In addition to the extremely high efficiency of genome editing of primordial germ cells, primordial germ cells genetically modified by genome editing have a high probability of differentiation into the germ line, so that genetically modified individuals were obtained with high probability. Is. In addition, conventional female primordial germ cells are weak in proliferating ability that can be killed by culturing for about 100 days, and it is considered that the genetically modified female primordial germ cells are also very weakly differentiated into the germline. Genetically modified female chimeric individuals could be obtained for the first time in the present invention using genome editing. Since the gene modification technology of the present invention enables highly efficient gene knockout, anti-disease against highly pathogenic avian influenza, etc. caused by virus receptor protein or sugar chain modification or deletion by destroying the target gene Development of sex poultry and changes or deletions of anti-hormone hormones, such as meat poultry that do not suppress food intake and grow in a short period of time, or low-growth pet poultry due to mutations or deletions of growth hormone genes It can also lead to efficient development.

By knocking in a foreign gene under an oviduct promoter such as ovomucoid or ovalbumin, foreign gene-derived protein can be highly expressed in the egg without being affected by silencing due to positional effects, which can lead to inexpensive protein production. . In addition, it is strongly expected that foreign gene-derived proteins can be expressed more efficiently than the conventional egg bioreactor technology.

Furthermore, female genetically modified G0 chimeras can be established by long-term culture and genetic modification of female primordial germ cell lines, and homozygous genetically modified individuals can be established in a short period of time by mating with male genetically modified G0 chimeras. Can do. Thereby, the establishment period of homo knockout poultry and knock-in poultry can be significantly shortened. Furthermore, by allowing mating of genetically modified G0 chickens (chimeric individuals), various combinations of genetically modified chickens (F1 and later) can be established as progenies significantly faster than before. In addition, because poultry have a WZ-type sex chromosome, it is possible to knock out genes on the W chromosome and knock in on the W chromosome, which was not possible only in females, and artificially phenotyped the sexual type It is possible to grant to.

Target sequence of chicken ovalbumin gene (2 sites, target sequences of OVATg1 and OVATg3). Uppercase letters indicate sgRNA recognition sites, adjacent underlined parts indicate PAM sequences Target sequence of chicken ovomucoid gene (4 sites, target sequences of OVMTg2, OVMTg3, OVMTg5, OVMTg6) Uppercase letters indicate sgRNA recognition sites and adjacent underlined parts indicate PAM sequences. OVMTg5 contains intron region (hatched area is intron / exon boundary) Example of ovalbumin gene disruption by CRISPR Uppercase (underlined) is sgRNA recognition site, adjacent box shows PAM sequence Deletion of mutated sequence is-(hyphen), mutation is capitalized 大文字 OVATg1 Met starts translation Indicates the site. Example of disruption of ovomucoid gene by CRISPR Uppercase (underlined) indicates sgRNA recognition site, adjacent box indicates PAM sequence では In the OVMTg5 sequence, the intron / exon boundary is indicated by / (slash) Mutation deletion part is-( Hyphens), mutations are capitalized Top: An example of a chicken in which the ovomucoid gene has been disrupted. The ovomucoid gene in the chick germ-derived chick (black) in the photograph is a single allele and lacks 5 bases in the Tg2 region of Fig. 2. The result of having analyzed the base sequence from the sense side and the antisense side of the region of the chicken genome is shown. Bottom: Ovomucoid gene mutation (F1 chicken) found in chicken individuals. A deletion of 1 to 31 bases was observed. Demonstration of knock-in by exogenous gene (EGFP donor construct) knock-in to the ovalbumin locus and genomic PCR. Primer 1 (P1) to Primer 8 (P8) correspond to the following sequences: P1: SEQ ID NO: 27, P2: SEQ ID NO: 29, P3: SEQ ID NO: 28, P4: SEQ ID NO: 26, P5: SEQ ID NO: 30, P6: Sequence No. 32, P7: SEQ ID NO: 33, P8: SEQ ID NO: 31 Nested As a result of PCR, an amplification product of the expected size is recognized only in the genome derived from knock-in primordial germ cells (PGCs) (photo, arrow) Examination of knock-in efficiency by foreign gene knock-in to the ovalbumin gene locus and genome quantitative PCR Primer 1 (P1) to Primer 3 (P3) correspond to the following sequences. P1: SEQ ID NO: 34, P2: SEQ ID NO: 35, P3: SEQ ID NO: 36 As a result of OVA (ATG) quantification, the donor construct was knocked in at 90% or more of ovalbumin loci in knockin primordial germ cells (PGCs). Also, as a result of OVA5 ', there is almost no random insertion Demonstration of knock-in by human interferon β gene knock-in to the ovalbumin locus and genomic PCR. Primer 1 (P1) to Primer 8 (P8) correspond to the following sequences: P1: SEQ ID NO: 27, P2: SEQ ID NO: 29, P3: SEQ ID NO: 41, P4: SEQ ID NO: 40, P5: SEQ ID NO: 30, P6: Sequence No. 32, P7: SEQ ID NO: 33, P8: SEQ ID NO: 31 Nested As a result of PCR, an amplification product of the expected size is recognized only in the genome derived from knock-in primordial germ cells (PGCs) (photo, arrow) Demonstration of human interferon beta gene knock-in to the ovalbumin locus in chimeric chicken sperm. The semen genome and knock-in cell (PCIFNKI # 4) genome of four chimeric chickens (411-414), one negative control chicken (416, NC) are represented by SEQ ID NOs: 30 and 31 (3'UTR), SEQ ID NO: 27 Amplified with primers of 40 (5'OVAp_out-IFN), SEQ ID NOS: 27 and 36 (5'OVAp_out-OVA (ATG). Bands appearing at the expected size are indicated by *. Positive controls at 411 and 412 A knock-in signal of the same relative intensity is observed. A chicken in which the human interferon β gene is knocked in at the ovalbumin gene locus. Photographs of progeny chickens (female) 411 and 412 in FIG. 7B and PCR analysis of the progeny blood-derived genome show that the IFN donor construct was knocked in at the ovalbumin locus. Wild-type (WT: negative control (NC)) chicken wing shaft (Shaft) -derived genome, knock-in progeny (KI) chicken wing shaft (Shaft) -derived genome and knock-in cell (KI PGC: positive control (PC))-derived genome Were amplified using primers of SEQ ID NOs: 30 and 31 (knock-in 3 ′ region), SEQ ID NOs: 27 and 40 (knock-in 5 ′ region), and SEQ ID NOs: 27 and 36 (endogenous ovalbumin). Bands that appear in the expected size are marked with *. In the 411, 412 progeny, a signal of the same pattern as the positive control was observed, and it can be determined that the IFN donor construct was knocked in at the ovalbumin locus of the progeny chicken. Target sequence of chicken ovalbumin gene (2 sites, target sequences of OVATg1 and OVATg2). The capital letter indicates the sgRNA recognition site, and the adjacent underline indicates the PAM sequence. Efficiency of gene knock-in to the chicken primordial germ cell ovalbumin locus. Since the efficiency of knock-in is not changed between introduction group 1 and introduction group 2, and the number of cells is larger in introduction group 2, the introduction method of introduction group 2 is more preferable. Introducing Group 2 and Introducing Group 3 are considered to have higher knock-in efficiency than Introducing Group 3, and the introducing method of Introducing Group 3 is more preferable. Human immunoglobulin gene knock-in to the ovalbumin locus and demonstration of knock-in by genomic PCR. Primer 1 (P1) to Primer 8 (P8) correspond to the following sequences: P1: SEQ ID NO: 27, P2: SEQ ID NO: 29, P3: SEQ ID NO: 44, P4: SEQ ID NO: 43, P5: SEQ ID NO: 30, P6: Sequence No. 32, 32 P7: SEQ ID NO: 33, P8: SEQ ID NO: 31 Nested As a result of PCR, an amplified product of the expected size is recognized only in the genome derived from knock-in primordial germ cells (PGCs) (photograph, arrow). Human collagen gene knock-in to the ovalbumin locus and demonstration of knock-in by genomic PCR. Primer 1 (P1) to Primer 8 (P8) correspond to the following sequences: P1: SEQ ID NO: 27, P2: SEQ ID NO: 29, P3: SEQ ID NO: 44, P4: SEQ ID NO: 43, P5: SEQ ID NO: 30, P6: Sequence No. 32, 32 P7: SEQ ID NO: 33, P8: SEQ ID NO: 31 Nested As a result of PCR, an amplified product of the expected size is recognized only in the genome derived from knock-in primordial germ cells (PGCs) (photograph, arrow). Sexual determination of cells by long-term culture of female primordial germ cells (PGCs) and genomic PCR. Sexual differentiation was possible by CHD (chromo-helicase-DNAasebinding protein) gene amplification, indicating that the cultured primordial germ cells were female karyotypes. Increase in the number of male primordial germ cells (about 4 months in culture), female primordial germ cells by this method (about 3 months in culture), and female primordial germ cells by conventional methods (with centrifugation at passage, about 40 days in culture) Comparison. After 8 days of culture, the cell numbers were 12.2 times, 4.0 times, and 0.9 times, respectively. Progeny derived from female primordial germ cells cultured by this method (arrows) and progeny derived from recipients of the same parent (back, 3 black feathers) Stable gene transfer into female primordial germ cells and establishment in the gonad (ovary) after early chicken embryo blood transplantation Example of allergen gene disruption in female primordial germ cell Uppercase (underlined) is sgRNA recognition site, adjacent box shows PAM sequence 欠失 Deletion of mutated sequence is-(hyphen), mutation is capitalized OVATg3 of ovalbumin Gene and OVMTg2 region of ovomucoid Demonstration of knock-in by exogenous gene (EGFP donor construct) knock-in to the ovalbumin locus in female primordial germ cells and genomic PCR. Primer 1 (P1) to Primer 8 (P8) correspond to the following sequences: P1: SEQ ID NO: 27, P2: SEQ ID NO: 29, P3: SEQ ID NO: 28, P4: SEQ ID NO: 26, P5: SEQ ID NO: 30, P6: Sequence No. 32, P7: SEQ ID NO: 33, P8: SEQ ID NO: 31 Nested As a result of PCR, an amplification product of the expected size is recognized only in the genome derived from knock-in primordial germ cells (PGCs) (photo, arrow) Human interferon β gene knock-in to the ovalbumin locus in female primordial germ cells and demonstration of knock-in by genomic PCR. Primer 1 (P1) to Primer 8 (P8) correspond to the following sequences: P1: SEQ ID NO: 27, P2: SEQ ID NO: 29, P3: SEQ ID NO: 41, P4: SEQ ID NO: 40, P5: SEQ ID NO: 30, P6: Sequence No. 32, P7: SEQ ID NO: 33, P8: SEQ ID NO: 31 Nested As a result of PCR, an amplification product of the expected size is recognized only in the genome derived from knock-in primordial germ cells (PGCs) (photo, arrow)

In the present invention, the genes of primordial germ cells in poultry are modified by genome editing.

Genome editing is a technology that uses a double-strand DNA break and its repair error to modify the gene. A nuclease that can cleave the target double-strand DNA and a DNA recognition component that binds or is complexed with the nuclease. Can be used. Examples of genome editing include ZFN (zinc finger nuclease), TALEN, and CRISPR. For example, FFN (nuclease) and zinc finger motif (DNA recognition component) are used in ZFN, FokI (nuclease) and TAL effector (DNA recognition component) are used in TALEN, and Cas9 (nuclease) and guide are used in CRISPR. RNA (gRNA, DNA recognition component) is used. The nuclease used for genome editing only needs to have nuclease activity, and in addition to the nuclease, DNA polymerase, recombinase, and the like can also be used.

Examples of poultry include chickens, quails, turkeys, ducks, geese, long-tailed birds, chabos, pigeons, ostriches, pheasants, and guinea fowls, and preferably chickens and quails.

The primordial germ cell may be male or female. Conventionally, genetic modification of female primordial germ cells has been considered difficult because cell culture can only be achieved for about 100 days. In the present invention, medium replacement is preferably performed under normal pressure or low gravity acceleration, preferably by centrifugation. By separating and culturing the supernatant without separation, female primordial germ cells were successfully cultured for a period exceeding 280 days.
Such long-term culture enabled production of adult female poultry (female genetically modified chimeric individuals) into which genetically modified primordial germ cells had been transplanted. As shown in FIG. 11B, male primordial germ cells have high proliferation ability even when centrifugation is performed at the time of medium exchange. However, female primordial germ cells hardly proliferate when subjected to centrifugation every time the medium is changed. Although it is substantially difficult to obtain an individual, the ability to grow can be maintained by suppressing the centrifugal operation at the time of medium exchange, and a chimeric individual can be obtained.

Poultry primordial germ cells such as chickens are floating cells and are cultured in the presence of feeder cells such as BRL cells and STO cells. When subcultured, for example, as described in Non-Patent Document 3, primordial germ cells are transferred to the centrifuge tube together with the medium every few days, and the cells are precipitated by centrifugation at 300 g for about 5 minutes and resuspended in the medium. It is common to seed cells afterwards. By using this method, male primordial germ cells can be passaged for a long time, but passage of female primordial germ cells is difficult (FIG. 11B). It is noted that the female primordial germ cells were established but became impossible to maintain beyond 109 days and 77 days, respectively. When maintenance is impossible, most of the female primordial germ cells have died. As a method for maintaining female primordial germ cells for a long period of time, it has been found that female primordial germ cells can be cultured over 280 days by appropriately performing a gentler medium recovery method. A milder recovery method means that the gravitational acceleration applied to female primordial germ cells at the time of passage is less than 300 g, preferably 270 g or less, more preferably 200 g or less, more preferably 100 g or less, particularly preferably 50 g or less, most preferably The culture is performed under a low gravitational acceleration of 1 g (normal pressure). In addition, the term “appropriately” indicates at least once a month, preferably at least once a week, and more preferably at least twice a month. In addition, a gravitational acceleration exceeding 300 g may be added to the female primordial germ cells. Female primordial germ cells are sensitive to the acceleration of gravity acceleration, and the acceleration of gravity acceleration is performed within a range where the proliferation ability can be maintained. For example, in the example, if the load of the gravitational acceleration (300 g) due to the centrifugal operation at the time of medium replacement is up to once a week, the proliferation ability of female primordial germ cells can be maintained. A person skilled in the art can refer to this and determine the load of gravity acceleration that can maintain the growth ability and the frequency thereof.

Genes modified by genome editing include ovalbumin, ovomucoid, ovomucin, ovotransferrin, ovoinhibitor, ovoglobulin, lysozyme and other in ovo proteins, especially egg white protein, pigment cell stimulating hormone, leptin, cocaine-amphetamine regulated transcript It encodes growth-inhibiting proteins such as growth hormones and their receptors, membrane proteins recognized by avian infectious viruses, and glycosylases that synthesize sugar chains (eg, sialyltransferases) Genes to be used. The function of these genes can be reduced or deleted by genome editing of the genes.

Gene function is lost by knockout by genome editing, or is reduced or lost by partial deletion. When editing at least one base of a gene is deleted or inserted by genome editing, the gene function may be lost due to frame shift (knockout). If no frame shift occurs, some amino acids are deleted (partial) Deletion), the function may be reduced or lost. Deletions and substitutions can also cause stop codons (knockout).

When knocking in an exogenous gene by genome editing, if the exogenous gene is knocked in to an in ovo protein locus such as ovalbumin, ovomucoid, ovo inhibitor, ovoglobulin, lysozyme, ovomucin, ovotransferrin, etc. Is preferable because an egg containing an exogenous gene expression product is obtained. As a protein that is an expression product of such an exogenous gene, various secretory proteins and peptides are considered, and an antibody (monoclonal antibody) or a fragment thereof (for example, scFv, Fab, Fab ′, F (ab ′) ₂ , Fv, single chain antibody, scFv, dsFv, etc.), enzyme, hormone, growth factor, cytokine, interferon, collagen, extracellular matrix molecule, functional polypeptide such as vaccine, agonistic protein, antagonistic protein, etc. It is done. The protein encoded by the exogenous gene is derived from a mammal, preferably a human, in the case of a physiologically active protein that can be a drug to be administered to humans. In addition, in the case of proteins that can be used industrially, such as protein A and the protein that constitutes the silk thread, proteins derived from any organism including microorganisms (bacteria, yeast, etc.), plants and animals, or artificial proteins Examples include exogenous genes encoding.

In addition, by editing genes such as fluorescent molecules under the control of cell-specific promoters and tissue-specific promoters by genome editing, fluorescent proteins are expressed only in specific cells and tissues. Experimental chicken embryos and individual chickens can be created.

Genomic editing includes zinc finger, TALEN, CRISPR, etc., TALEN and CRISPR are preferred, and CRISPR is more preferred. Genome editing methods have been developed one after another, and the present invention is not limited to these. Any genome editing method developed in the future can be used in the present invention.

When knock-in is performed by genome editing, it is preferable to stably integrate a drug resistance gene together with a useful exogenous gene into the genome, thereby selecting a primordial germ cell knocked-in. Drug resistance genes include neomycin resistance gene (Neo ^r ), hygromycin resistance gene (Hyg ^r ), puromycin resistance gene (Puro ^r ), blasticidin resistance gene (blast ^r ), zeocin resistance gene (Zeo ^r ), etc. Neomycin resistance gene (Neo ^r ) or puromycin resistance gene (Puro ^r ) is preferable.

When genome editing is performed and gene function is lost by knockout, or is reduced or eliminated by partial deletion, the above drug resistance genes are introduced into primordial germ cells at the time of gene introduction when genome editing is performed, and selected based on drug resistance genes It is preferable to do. Introduction of drug resistance gene and drug selection may be stable or transient, and transient is desirable in the case of knockout or partial deletion. Drug resistance genes such as those described above can be mentioned, puromycin resistance gene (Puro ^r) or zeocin resistance gene (Zeo ^r) are preferred. The drug resistance gene may be in the form independent of the zinc finger, TALEN, or CRISPR plasmid, or may be incorporated into the plasmid, and the drug resistance gene is preferably incorporated into the plasmid for genome editing.

In one embodiment, genetically modified poultry can be produced from genetically modified poultry primordial germ cells obtained by the genetic modification method of the present invention according to a conventional method. The specific procedure is shown below.

The genetically modified primordial germ cells are transplanted into the blastoderm, blood or gonad region of the recipient early embryo. Preferably, several hundred to several thousand cells are transplanted by microinjection into the blood stream at a time before and after the start of blood circulation for about 2 to 3 days after incubation. In addition, the recipient's endogenous primordial germ cells may be inactivated in advance by a drug or ionizing radiation, or the number may be reduced before transplantation. Incubation of the transplanted embryo is continued according to a conventional method, and the transplanted individual is hatched. The transplanting and hatching operations may be system culture that includes eggshell changes, or may be a window opening method that does not change eggshells. The hatched individual can be sexually matured as a living body (chimeric individual) by normal breeding. By breeding this with a wild-type or genetically modified individual or a genetically modified chimeric individual, a poultry having a genetic modification derived from a transplanted cell can be produced as a progeny. The genome-edited primordial germ cells obtained in the present invention have a high proliferation ability and become a large number of highly fertilized sperm or eggs in a chimeric individual. At this time, in order to increase efficiency, we investigated the frequency of genetic modification contained in the gamete genome, evaluated the contribution rate of transplanted cells, performed a mating test, and performed a determination based on the progeny feather color. May be. A homozygous genetically modified poultry can be obtained by mating female chimeric poultry transplanted with genetically modified female primordial germ cells and male chimeric poultry transplanted with male primordial germ cells. In addition, not only within poultry individuals, but if technologies such as in vitro differentiation of primordial germ cells into germ cells are developed in the future, genetically modified poultry will be produced by artificial insemination and microinsemination using this. can do.
In FIG. 2, FIG. 4A, FIG. 4B, and FIG. 13, the PAM sequence of OVTg2 is “agg”, but there are two types of sequences corresponding to OVMg2 of chicken ovalbumin in the NCBI database, and the sequence of OVTg2 is TTTCCCAACGCTACAGACA (T or a) gg. The present invention encompasses all such polymorphisms.

In another embodiment of the present invention, genome editing is performed by infecting various embryos with a viral vector or injecting a plasmid vector as a liposome complex into early embryo blood without going through primordial germ cell culture. Alternatively, endogenous primordial germ cells may be genetically manipulated to establish chimeric individuals and recombinant progeny. Primordial germ cells obtained by genome editing have high gene modification efficiency and have a sufficiently high reproductive ability to obtain a recombination progeny or gene modification progeny of poultry, which is also useful in this embodiment. In this embodiment, genetic modification of (endogenous) primordial germ cells is possible without culturing primordial germ cells.

Examples of virus vectors used for gene manipulation by genome editing include retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, and lentivirus vectors. These viral vectors can be used for genome editing of cultured primordial germ cells or endogenous primordial germ cells.

For example, to modify endogenous primordial germ cells using genome editing, a viral vector that expresses a nuclease or sgRNA that recognizes and cleaves any target sequence using a genome editing viral vector sold by each company. , And make the form infectable by packaging, and administer genomes in primordial germ cells by administering to the place where primordial germ cells exist such as blastoderm, blood and gonad area of early poultry embryos. It is possible to obtain genetically modified individuals and genetically modified products. Commercially available viral vectors for genome editing are sold by many companies in Japan and overseas.For example, Takara's `` AAVpro (registered trademark) CRISPR / Cas9 Helper Free System (AAV2) '' and lentivirus using adeno-associated vectors Examples include "Lentiviral CRISPR / Cas9 System" by System Biosciences, LLC. When the genetic modification is knock-in, a viral vector necessary for genome editing and a viral vector, plasmid or the like containing the knocked-in gene can be used in combination.

In addition, genome editing plasmids and donor constructs that do not use or are combined with viral vectors are made to be permeable to cell membranes such as liposome complexes, and primordial germ cells such as blastoderm, blood and gonad areas of early poultry embryos It is possible to perform genome editing in primordial germ cells and administer gene-modified individuals and gene-modified products to progenies.

Hereinafter, the present invention will be described in more detail based on examples.

Example 1
(1) Genome editing using chick male primordial germ cells (1-1) Gene construction for ovalbumin (OVA) and ovomucoid (OVM) knockout Targeting ovalbumin and ovomucoid gene using chick male primordial germ cell line The gene was disrupted by the CRISPR method. As shown in FIG. 1 (ovalbumin) and FIG. 2 (ovomucoid), destruction of target sites at 2 sites (OVATg1, OVATg3) and 4 sites (OVMTg2, OVMTg3, OVMTg5, OVMTg6) was attempted.

A CRISPR plasmid was constructed by targeting the target sequences (two locations) of the ovalbumin gene shown in FIG.

First, oligo DNAs represented by SEQ ID NO: 6 and SEQ ID NO: 7 were synthesized using SEQ ID NO: 5 (OVATg3) as a target, and the 5 ′ end was phosphorylated using T4 Polynucleotide Kinase, and then the mixture of both was heated to 98 ° C. It annealed by heating and cooling slowly to room temperature. This DNA fragment was inserted into the BbsI cleavage site of the plasmid px330-Puro ^r inserting the puromycin resistance gene unit into the NotI site SEQ ID NO: 8 of the plasmid px330 (AddGENE, ^{USA) (px330-Puro r -OVATg3)} . In addition, a plasmid was constructed in which the puromycin resistance gene unit of px330-Puro ^r -OVATg3 was replaced with the zeocin resistance gene unit of SEQ ID NO: 124 (px330-Zeo ^r -OVATg3). Furthermore, a plasmid was constructed in which the puromycin resistance gene unit of px330-Puro ^r -OVATg3 was replaced with the neomycin resistance gene unit represented by SEQ ID NO: 4 (px330-Neo ^r -OVATg3). In addition, oligo DNAs shown in SEQ ID NO: 2 and SEQ ID NO: 3 were synthesized, phosphorylated and annealed targeting SEQ ID NO: 1 (OVATg1), and the neomycin resistance gene unit shown in SEQ ID NO: 4 was inserted into the NotI site of plasmid px330. The inserted plasmid px330-Neo ^r was inserted into the BbsI cleavage site (px330-Neo ^r -OVATg1).

A CRISPR plasmid was constructed by targeting the target sequence (four locations) of the ovomucoid gene shown in FIG. Oligo DNAs represented by SEQ ID NO: 10 and SEQ ID NO: 11 were synthesized targeting SEQ ID NO: 9 (OVMTg2), phosphorylated, annealed, and inserted into the BbsI cleavage site of plasmid px330-Puro ^r (px330-Puro ^r − OVMTg2). Further, plasmids were constructed in which the puromycin resistance gene unit of px330-Puro ^r -OVMTg2 was replaced with the zeocin resistance gene unit of SEQ ID NO: 124 and the neomycin resistance gene unit represented by SEQ ID NO: 4 (px330-Zeo ^r -OVMTg2 and px330-Neo ^r -OVMTg2). Furthermore, SEQ ID NO: 12 (OVMTg3) as a target, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 (OVMTg5) as a target, SEQ ID NO: 16 and SEQ ID NO: 17, and SEQ ID NO: 18 (OVMTg6) as a target, SEQ ID NO: 19 and SEQ ID NO: The oligo DNAs indicated by No. 20 were synthesized, phosphorylated, annealed, and each DNA fragment was inserted into the BbsI cleavage site of the plasmid px330-Neo ^r (px330-Neo ^r -OVMTg3, px330-Neo ^r -OVMTg5 px330-Neo ^r -OVMTg6).

(1-2) Ovalbumin and ovomucoid gene knockout (1-2-1) Efficiency of mutation introduction by transient drug selection Chicken male primordial germ cells collected from the blood of the lateral primus rock male embryo and established ( The above gene (plasmid) was transiently introduced into Non-Patent Document 3). 1 × 10 ⁵ to 5 × 10 ⁵ male primordial germ cell lines were washed with PBS, suspended in OPTI-MEM, and 1.6 μg px330-Puro ^r using 3 μl Lipofectamine 2000 (Life Technologies, USA) -OVATg3 was introduced. Specifically, Lipofectamine 2000 and plasmid were mixed in 80 μl OPTI-MEM, mixed with the male primordial germ cell line and allowed to stand at room temperature for about 5 minutes, and then 500 μl of medium containing no antibiotics was added, and the mixture was incubated at 37 ° C. And allowed to stand for about 1 to 4 hours, and then seeded on feeder cells. Between 2 and 4 days after gene transfer, puromycin (InvivoGen, USA) was added at a final concentration of 1 μg / ml, and this was washed and removed, followed by culturing for 1 to 2 weeks. After the culture, the cells were collected and the genomic DNA was extracted. Then, a partial region of the ovalbumin gene was amplified by PCR using the oligo DNA primers represented by SEQ ID NO: 21 and SEQ ID NO: 22, and the TA vector (pGEM-T Easy , Promega, USA), and the genomic nucleotide sequence of the region containing SEQ ID NO: 5 (OVATg3) was analyzed. In all 12 clones analyzed (100%), deletion of the gene was observed in the region containing the ovalbumin gene SEQ ID NO: 5 (OVATg3). On the other hand, although no drug selection was performed as a control group, gene deletion was limited to 3 out of 45 clones (6.7%). Many of these mutations included frame shifts during translation. Further, similarly Transgenic px330-Zeo ^r -OVATg3, between 2-4 days after the gene introduction, final concentration 50 [mu] g / ml Zeocin (InvivoGen, USA) were cultured in the presence, after washing, 1-2 weeks As a result of conducting the same gene analysis using the cells cultured in the above-described manner, 11 clones (92%) out of 12 analyzed clones showed gene deletion. FIG. 3 shows examples of gene mutations observed in the region containing OVATg3.

Next, the same analysis was performed targeting the ovomucoid gene. The px330-Puro ^r -OVMTg2 using Lipofectamine 2000 to ^{1 × 10 5 ~ 5 × 10} 5 cells of the male primordial germ cell line similar to the above 1.6μg gene transfer between 2-4 days after introduction, puromycin Was added at a final concentration of 1 μg / ml. After puromycin has been removed by washing, culture is performed for 1 to 2 weeks, cells are collected, genomic DNA is extracted, and one of the ovomucoid genes is obtained by PCR using oligo DNA primers represented by SEQ ID NO: 23 and SEQ ID NO: 24. A partial region was amplified, subcloned into a TA vector, and the genomic base sequence of the region containing SEQ ID NO: 9 (OVMTg2) was analyzed. In 21 clones (91%) out of 23 clones analyzed, deletion of the gene was observed in the region containing SEQ ID NO: 9 (OVMTg2) of the ovomucoid gene. On the other hand, although no drug selection was performed as a control group, 0 genes (0%) were found in 24 clones. Further, similarly Transgenic px330-Zeo ^r -OVMTg2, cultured in the zeocin presence Matsui concentration 50 [mu] g / ml of 2-4 days after the introduction, in 1-2 weeks of culture cells were washed and analyzed 11 Mutations were found in 10 clones (91%). FIG. 4A shows an example of gene mutation observed in the region containing OVMTg2. These facts indicate that in gene editing of poultry primordial germ cell genes, introduction of drug resistance genes and transient selection by drugs can significantly increase the efficiency of mutations, especially drugs by puromycin resistance genes and puromycin. It shows that high mutation efficiency can be obtained by selection or drug selection with zeocin resistance gene and zeocin.

(1-2-2) Mutation introduction into various regions of ovalbumin and ovomucoid Introducing px330-Neo ^r -OVATg1 targeting the region including the translation start point of the ovalbumin gene into chicken primordial germ cells using the method described above Then, select neomycin (G418 disulfate, Nacalai Tesque, Japan) at a final concentration of 0.5 mg / ml for 2-4 days after introduction, and use the same method as 1-2-1 to determine the genomic base sequence containing the target region. Analyzed. As shown in FIG. 3, mutations including deletion and substitution of the start codon were confirmed.

In addition, mutations were introduced targeting various regions of the ovomucoid gene. The ovomucoid protein has three domains (domain 1 to domain 3) that are considered to have strong allergenicity. The purpose px330-Neo ^r -OVMTg3 lacking domains 1 and later, leaving the domain 1, domain 2 and later px330-Neo in lacking purposes ^r -OVMTg5, by px330-Neo ^r -OVMTg6 respective to the method described above After gene introduction into chicken primordial germ cells, neomycin was selected in the same manner as described above, and the genomic nucleotide sequence was analyzed. As shown in FIG. 4A, gene deletion, substitution, and insertion occurred in the region containing the target site of the allergen gene ovomucoid as shown in FIG. 4A, and mutations including frameshift and termination during translation were confirmed. As described above, gene editing technology using primordial germ cells enables gene knockout caused by mutations in various target sites.

(1-3) Establishment of genome editing chickens px330-Puro ^r -OVMTg2 to Barred Plymouth Rock species primordial germ cells in the manner described in (1-2-1) was introduced gene, culturing the drug selected cells, Transplantation was performed by microinjection into the blood of a white Leghorn 2.5 day embryo (recipient embryo). Prior to transplantation, fertilized eggs before incubation were irradiated with 5 Gy or 6 Gy for the purpose of reducing the number of primordial germ cells in the recipient embryo. The ionizing radiation was performed by gamma irradiation using a gamma cell 40 (Canadian Atomic Energy Corporation).

After 2.5 days of incubation, open a window about 2cm in diameter on the tip of the egg to expose the embryo, and about 1000-5000 drug-selected cells (1-2 μl) in the recipient embryo blood from Hamburger Hamilton stages 13-15 (Suspended in PBS) was transplanted using a fine glass needle. After sealing the window with cellophane tape, it was incubated and incubated at a temperature of 38.5 ° C and a humidity of 60-80% (chimeric chick (G0)). Eight male chimeric chicks were sexually matured and semen collected. After genomic DNA is extracted from semen, a partial region of the ovomucoid gene is amplified by PCR using the oligo DNA primers shown in SEQ ID NO: 23 and SEQ ID NO: 24, subcloned into a TA vector, and SEQ ID NO: 9 (OVMTg2) The genomic base sequence of the region containing was analyzed. Crossing chimera chickens # 372 and # 376 with high frequency mutations (both 10 of the 11 subcloned clones with ovomucoid gene mutations) with wild-type females with lateral Plymouth Rock Plymouth Rock, We found 11 ovumcoid mutant chickens (chicks) out of 19 out of 19 progenies and 14 out of 14, respectively. An example of ovomucoid gene mutation is shown in the upper part of FIG. 4B. In this individual, a deletion of 5 bases that causes mutation is observed from directly under the signal peptide of the ovomucoid protein, and one allele has a frameshift mutation of the ovomucoid gene. In addition, examples of typical mutations (gene deletions) observed mainly in the target region of the ovomucoid genome are shown in the lower part of FIG. 4B. We obtained several male and female individuals of ovalbumin hetero knockout that produce frameshift mutations directly below the signal peptide of the ovomucoid protein represented here, and crossing these individuals after sexual maturation can produce homo knockout chickens of ovomucoid. Is possible.

Example 2
(1) Gene knock-in to ovalbumin gene locus Gene knock-in by genome editing was performed using primordial germ cells derived from male chicken. A donor construct shown in SEQ ID NO: 25 (EGFP donor construct) was prepared for the purpose of inserting a foreign gene (EGFP) at the translation start point of the ovalbumin gene. This EGFP donor construct is composed of about 2.8 kb at the 5 ′ side of the ovalbumin translation start point, EGFP gene, drug resistance gene unit (PGK-Puro ^r ), and about 3.0 kb at the 3 ′ side of the ovalbumin translation start point. This donor construct was inserted into a plasmid pBlue ScriptII (SK +) (Stratagene, currently Agilent Technologies, USA) to obtain a pBS-EGFP donor. Similarly to Example 1, 0.8 μg of px330-Neo ^r -OVATg1 and 0.8 μg of pBS-EGFP donor were simultaneously introduced into 1 × 10 ⁵ to 5 × 10 ⁵ primordial germ cell lines using Lipofectamine 2000. After 3 days, puromycin was added at a final concentration of 1 μg / ml. The medium was appropriately changed, and cells proliferating in the presence of puromycin at a final concentration of 1 μg / ml were collected to prepare genomic DNA.

Genomic PCR confirmed that the donor construct was knocked into the ovalbumin locus. For the 5 ′ region, PCR using primers for the foreign gene of the donor construct and the 5 ′ region of ovalbumin not included in the donor construct was performed as follows. PCR was performed using an antisense primer for EGFP shown in SEQ ID NO: 26 and a sense primer for a region about 3.0 kb on the 5 ′ side of the ovalbumin translation start point shown in SEQ ID NO: 27. PCR was performed using the antisense primer for EGFP shown and the sense primer for the region about 2.85 kb 5 ′ from the ovalbumin translation start point shown in SEQ ID NO: 29 and not included in the donor construct (nested PCR). As shown in FIG. 5, when a donor construct is introduced together with px330-Neo ^r -OVATg1, and a drug-selected primordial germ cell (knock-in PGCs) -derived genome is used as a template, about 2.9 expected when the donor construct is inserted. An amplification product is observed at the position k, whereas when a primordial germ cell-derived genome (control PGCs) not subjected to control gene transfer is used as a template, no amplification product is observed.

Similarly, the 3 ′ region was confirmed by genomic PCR using primers for the foreign gene of the donor construct and the 3 ′ region of ovalbumin not included in the donor construct. Perform PCR using the sense primer for the drug resistance gene unit shown in SEQ ID NO: 30 and the antisense primer for the region about 3.4 kb on the 3 ′ side of the ovalbumin translation start point shown in SEQ ID NO: 31, and then sequence the amplified product. PCR was performed using a sense primer for the drug resistance gene unit shown in No. 32 and a sense primer for a region not included in the donor construct at about 3 kb 3 ′ of the ovalbumin translation start point shown in SEQ ID No. 33 (nested) PCR). As shown in FIG. 5, when a donor construct is introduced together with px330-Neo ^r -OVATg1, and a drug-selected primordial germ cell (knock-in PGCs) -derived genome is used as a template, about 3.4 expected when the donor construct is inserted. An amplification product is observed at the position k, whereas when a primordial germ cell (control PGCs) -derived genome that has not been subjected to control gene transfer is used as a template, no amplification product is observed. From the above, it is considered that there are cells in which a donor construct containing a foreign gene part is knocked in at the ovalbumin gene locus in the cell population selected by the drug.

(2) Assay of knock-in efficiency Next, it was assayed to what extent the cells in which the donor construct was knocked into the ovalbumin gene locus were present in the drug-selected cell group. Genomic DNAs were prepared from drug-selected cell groups and primordial germ cells (control PGCs) that had not been transfected. Using this as a template, quantitative PCR (OVA5 ') with primers shown in SEQ ID NO: 34 and SEQ ID NO: 35, quantitative PCR (OVA (ATG)) with primers shown in SEQ ID NO: 34 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: Quantitative PCR (GAPDH (glyceraldehyde-3-phosphate dehydrogenase)) with the primer shown in 38 was performed. The reaction was analyzed by 7300 Real Time PCR system (Applied Biosystems, USA) using THUNDERBIRD qPCR Mix (Toyobo, Japan). As schematically shown in FIG. 6, the PCR product of OVA5 ′ can be amplified using any of the ovalbumin gene not knocked in, the randomly inserted donor construct, and the knocked-in donor construct as a template. On the other hand, PCR products of OVA (ATG) use only the ovalbumin gene that has not been knocked in as a template, and amplification using a randomly inserted donor construct or a knocked-in donor construct as a template hardly occurs because of the long amplification region. . GAPDH was used as an internal standard for genomic quantity. FIG. 6 shows a graph of quantitative PCR results corrected by the internal standard. The OVA 5 'PCR product is not significantly different in the drug selected cell population genome and the control primordial germ cell genome, whereas the OVA (ATG) PCR product is one-tenth of the control in the drug selected cell population genome. It is as follows. The fact that there is no significant difference in the PCR product of OVA5 ′ indicates that random insertion other than the knock-in of the donor construct is below the detection limit. On the other hand, the fact that the OVA (ATG) PCR product was one-tenth or less of the control in the drug-selected cell group genome indicates that the donor construct was knocked in in 90% or more of the ovalbumin gene in this genome. Show. That is, it is determined that 90% or more of the ovalbumin gene is replaced by the knock-in construct in the primordial germ cell group selected by the drug.

Example 3
Human interferon gene knock-in (1) Knock-in to primordial germ cell establishment and establishment of knock-in chimera chicken Donor construct (IFNβ) in which human interferon β gene shown in SEQ ID NO: 39 is introduced instead of EGFP in the EGFP donor construct of Example 2 above Donor construct) was prepared. This donor construct is composed of about 2.8 kb 5 ′ from the ovalbumin translation start point, human interferon β gene, drug resistance gene unit (PGK-Puro ^r ), and about 3.0 kb 3 ′ from the ovalbumin translation start point. This donor construct was inserted into the plasmid pBlue ScriptII (SK +) to obtain a pBS-IFNβ donor. By knocking in male chick primordial germ cells and selecting with puromycin in the same manner as the above pBS-EGFP donor, PCR was performed using the genome of the selected cells as a template. On the 5 ′ side, after PCR with the primer of SEQ ID NO: 27 and the antisense primer for interferon β shown in SEQ ID NO: 40, the primer of SEQ ID NO: 29 and the antisense primer for interferon β shown in SEQ ID NO: 41 are applied to the amplified product. PCR was performed using (nested PCR). On the 3 ′ side, after PCR using the primers shown in SEQ ID NO: 30 and SEQ ID NO: 31 in the same manner as when knocking in the EGFP donor, nested PCR was performed using the primers shown in SEQ ID NO: 32 and SEQ ID NO: 33 on the amplified product. Went. As shown in FIG. 7A, knock-in to the ovalbumin gene in primordial germ cells was observed even when an IFNβ donor was used in the same manner as the EGFP donor. Further, the knock-in efficiency test was carried out by quantitative PCR as in Example 2- (2). As a result, it was determined that 85% or more of the ovalbumin gene was replaced by the IFNβ donor construct.

Primordial germ cells containing cells into which this IFNβ donor construct was knocked in were transplanted into recipient embryos by the same method as in (1-3) and then hatched to obtain four male chimeric chickens (# 411 to # 414) . Semen was collected from these, and genomic DNA was collected, and then the primers of SEQ ID NO: 30 and SEQ ID NO: 31 (amplified on the 3 ′ side of interferon knocked into the ovalbumin gene), the primers of SEQ ID NO: 27 and SEQ ID NO: 40 (ovobo) PCR was performed using the primers of SEQ ID NO: 27 and SEQ ID NO: 36 (amplified ovalbumin that was not knocked in), respectively (amplification of the interferon knocked into the albumin gene) (FIG. 7B). In chimera chickens # 411 and # 412, signals clearly showing interferon knock-in to the ovalbumin locus are observed on both the 3 ′ side and the 5 ′ side, and in particular, # 411 is observed with a signal intensity comparable to the transplanted parent strain. Therefore, it is considered that cells in which interferon is knocked in to the same degree as the parent strain exist in the sperm. *

Chimera chickens # 411 and # 412 were crossed with female wild-type chickens (lateral primus rock species) to obtain 28 progeny and 19 progeny, respectively. After collecting the wing shaft from this progeny, collecting genomic DNA, the primers of SEQ ID NO: 30 and SEQ ID NO: 31 (amplifying the 3 ′ side of interferon knocked into the ovalbumin gene), SEQ ID NO: 27 and PCR was performed using primer No. 40 (amplification of 5 ′ side of interferon knocked into ovalbumin gene) and primer of SEQ ID No. 27 and SEQ ID No. 36 (amplification of ovalbumin not knocked in). In addition, the same PCR was performed using a wild-type wing shaft-derived genome (negative control (NC)) and a transplanted interferon donor vector knock-in primordial germ cell-derived genome (positive control (PC)). In 8 out of 28 progenies derived from # 411 and 5 out of 19 out of # 412 progeny, the signals clearly showing interferon knock-in to the ovalbumin locus similar to the positive control are respectively 3 'and 5' Both were recognized. FIG. 7C shows the PCR product electrophoresis images of the progeny from # 411 (female) and the progeny from # 412 (female), respectively. Based on this, it is determined that an interferon donor vector is knocked in at the ovalbumin locus in these progeny female chickens.

(2) Improvement of knock-in efficiency We investigated the improvement of gene knock-in efficiency. First, was prepared IFN beta-Neo donor constructs to replace the drug resistance units of interferon β donor construct described above PGK-Puro ^r from SV40Pe-Neo ^r (SEQ ID NO: 125). This donor construct is composed of about 2.8 kb on the 5 ′ side of the ovalbumin translation start point, human interferon β gene, drug resistance gene unit (SV40Pe-Neo ^r ), and about 3.0 kb on the 3 ′ side of the ovalbumin translation start point. This donor construct was inserted into the plasmid pBlue ScriptII (SK +) to obtain a pBS-IFNβ-Neo donor. Next, a plasmid px330-Puro ^r -OVATg1 was constructed in which the neomycin resistance unit of px330-Neo ^r -OVATg1 was replaced with the puromycin resistance unit of SEQ ID NO: 8. As shown in FIG. 8A, a plasmid for CRISPR was constructed by targeting the target sequence OVATg2 (SEQ ID NO: 126) of ovalbumin partially overlapping with OVATg1. Oligo DNAs respectively represented by SEQ ID NO: 127 and SEQ ID NO: 128 were synthesized, phosphorylated in the same manner as in Example 1-1, annealed, the DNA fragment was inserted into the BbsI cleavage site of px330, and SEQ ID NO: at the NotI site. 8 plasmid px330-Puro ^r -OVATg2 inserting the puromycin resistance units were constructed. After preparing about 5 × 10 ⁵ primordial germ cell lines and dividing into 3 parts, 0.8 μg of px330-Neo ^r -OVATg1 and pBS-IFNβ donor (by using Lipofectamine 2000 as in Example 2- (1) ( 0.8 [mu] g to have a puromycin resistance gene units) (0.8 [mu] g to introduce group 1) or px330-Puro ^r -OVATg1, the pBS-IFN beta-Neo donor 0.8 [mu] g (transfection group 2) or px330-Puro ^r -OVATg2 0.8 μg and 0.8 μg of pBS-IFNβ-Neo donor (introduction group 3) were introduced at the same time, and (introduction group 1) was administered puromycin at a final concentration of 1 μg / ml after 3 days after introduction as in Example 3- (1). Added at. On the other hand, (introduction group 2) and (introduction group 3) were cultured in the presence of puromycin at a final concentration of 1 μg / ml for 2 to 4 days after gene introduction, as in Example (1-2-1). After washing, neomycin with a final concentration of 0.5 mg / ml was added and cultured. When the number of cells in each introduction group was measured 24 days after introduction, 2 × 10 ⁴ in

introduction group

1 and 1 × 10 ⁵ drug-resistant cells were observed in

introduction groups

2 and 3. In addition, after collecting cells from each group and preparing genomic DNA, the primers of SEQ ID NO: 30 and SEQ ID NO: 31 (on the 3 ′ side of the interferon knocked into the ovalbumin gene) were prepared in the same manner as in Example 3- (1). Amplification), using the primers of SEQ ID NO: 27 and SEQ ID NO: 40 (amplify the 3 ′ side of the interferon knocked into the ovalbumin gene), and the primers of SEQ ID NO: 27 and SEQ ID NO: 36 (amplify the non-knocked ovalbumin) PCR was then performed (FIG. 8B). Since there is no significant difference in the ratio of PCR signal intensity between

introduction groups

1 and 2, it is considered that the target cells can be prepared more quickly by selecting neomycin after short-term selection with puromycin from introduction group 2. . Introduced group 3 has almost no ovalbumin not knocked in compared to introduced group 2, and therefore, more efficient knock-in is considered to have occurred. Based on the above, targeting the OVATg2 sequence, inserting the puromycin resistance gene into the CRISPR construct, inserting the neomycin resistance gene into the donor construct, introducing into the primordial germ cell, selecting with puromycin, and then selecting with neomycin Therefore, it is considered that knock-in of foreign genes to the ovalbumin locus can be performed quickly and efficiently.

Example 4
Human antibody gene knock-in A donor construct (immunoglobulin donor construct) in which the human immunoglobulin gene shown in SEQ ID NO: 42 was introduced instead of EGFP in the EGFP donor construct of Example 2 above was prepared. This donor construct is about 2.8 kb downstream of the ovalbumin translation start point, egg white lysozyme signal peptide, human immunoglobulin heavy chain, furin protein cleavage target sequence, 2A self-processing peptide, egg white lysozyme signal peptide, human immunoglobulin It arranged connecting a gene encoding the light chain gene, respectively, a drug resistance gene unit (PGK-Puro ^r), and a 3 'side about 3.0kb ovalbumin translation initiation. This donor construct is transcribed and translated to express an antibody protein composed of immunoglobulin heavy and light chains.

An immunoglobulin donor construct was inserted into the plasmid pBlue ScriptII (SK +) to obtain a pBS- IgG (Hc + Lc) donor. By knocking in male chick primordial germ cells and selecting with puromycin in the same manner as the above pBS-EGFP donor and pBS-IFNβ donor, PCR was performed using the selected cell genome as a template. The 5 ′ side was subjected to PCR using the primer of SEQ ID NO: 27 and the antisense primer for the egg white lysozyme signal peptide shown in SEQ ID NO: 43, and then the primer for SEQ ID NO: 29 and the egg white lysozyme signal peptide shown in SEQ ID NO: 44 to the amplified product. PCR was performed using antisense primers (nested PCR). The 3 ′ side is the primer shown in SEQ ID NO: 32 and SEQ ID NO: 33 for the amplified product after PCR using the primers shown in SEQ ID NO: 30 and SEQ ID NO: 31 as in the case of knocking in the EGFP donor and pBS-IFNβ donor. Was used for nested PCR. As shown in FIG. 9, knocking-in to the ovalbumin gene in primordial germ cells was observed even when an immunoglobulin donor was used. Further, as a result of quantitative PCR similar to Example 2- (2), it is determined that 90% or more of the ovalbumin gene has been replaced by the immunoglobulin donor construct.

Example 5
Human collagen gene knock-in A donor construct (collagen donor construct) was prepared by introducing the human type I collagen gene shown in SEQ ID NO: 45 instead of EGFP in the EGFP donor construct of Example 2 above. This donor construct is about 2.8 kb downstream of the ovalbumin translation start point, egg white lysozyme signal peptide, human type I collagen α1 chain (COLLAGEN1A1), furin protein cleavage target sequence, 2A self-processing peptide, egg white lysozyme signal peptide , arranged by connecting genes each encoding a human type I collagen α2 chain (COLLAGEN1A2) gene, a drug resistance gene unit (PGK-Puro ^r), it consists of three 'side about 3.0kb ovalbumin translation initiation Yes. This donor construct is transcribed and translated to express type I collagen protein consisting of human type I collagen α1 and α2 chains.

A collagen donor construct was inserted into the plasmid pBlue ScriptII (SK +) to obtain a pBS-COL1 (A1 + A2) donor. Select pBS-COL1 (A1 + A2) donor with puromycin after knock-in to male chicken primordial germ cells by the same method as the above pBS-EGFP donor, pBS-IFNβ donor, pBS-IgG (Hc + Lc) donor PCR was performed using the genome of the prepared cells as a template. On the 5 ′ side, as with the pBS-IgG (Hc + Lc) donor, after PCR with the primer of SEQ ID NO: 27 and the antisense primer for the egg white lysozyme signal peptide shown in SEQ ID NO: 43, the amplified product of SEQ ID NO: 29 PCR was performed using the primer and an antisense primer for the egg white lysozyme signal peptide shown in SEQ ID NO: 44 (nested PCR). 3 'side is the same as when knocking in the above EGFP donor, pBS-IFNβ donor, pBS-IgG (Hc + Lc) donor and after PCR using the primers shown in SEQ ID NO: 30 and SEQ ID NO: 31, Nested PCR was performed using the primers shown in SEQ ID NO: 32 and SEQ ID NO: 33. As shown in FIG. 10, knock-in to the ovalbumin gene in primordial germ cells was observed even when a collagen donor was used. Further, as a result of quantitative PCR similar to Example 2- (2), it was determined that 90% or more of the ovalbumin gene was replaced by the collagen donor construct.

Example 6
Cultivation of female primordial germ cells Isolation and culture of primordial germ cells from embryonic blood of highline Maria chick 2-3 days were performed according to Non-patent Document 3. Gender was determined by PCR using the primers shown in SEQ ID NO: 46 and SEQ ID NO: 47 for the CHD (chromo-helicase-DNA binding protein) gene and using the collected chicken embryo cell-derived genome as a template.

During passage, primordial germ cells in the medium were dispersed and collected using a pipette, and seeded on feeder cells (BRL cells). Prior to cell collection, a part of the upper layer of the medium (usually about 50 to 75%) was gently removed using an aspirator or pipette, and the medium was changed by adding a new medium. Primordial germ cells are suspension cells, but most cells accumulate in the lower part of the culture dish in culture under 1 g (normal pressure), so this method leaves most of the primordial germ cells and maintains the cells. However, it is possible to change most of the medium. In addition, if you do not want to lose cells as much as possible when replacing the medium, or if you want to completely replace the medium (removal of selective drugs or serum components during gene transfer), collect primordial germ cells together with the medium. Centrifugation was carried out at 300 g for about 5 minutes in the same manner as in 3. However, it was usually decided to perform passage without centrifugation at least once a week. By performing such passage, female primordial germ cells could be cultured for a long period of time. FIG. 11 shows female primordial germ cell images on the 100th day and the 230th day after the start of the culture, and it is possible to perform the culture while maintaining the spherical and floating characteristics. The cultured cells are female primordial germ cells. The CHD gene was amplified by PCR using the primers shown in SEQ ID NO: 46 and SEQ ID NO: 47 using the cultured cell-derived genome on day 100 of culture as a template. Confirmed. When CHD on the sex chromosome is amplified using the primers shown in SEQ ID NO: 46 and SEQ ID NO: 47, two products of about 400 bp and 600 bp are produced in the female genome and one product of 600 bp in the male genome. As shown in FIG. 11, the female karyotype was confirmed using a genome derived from female primordial germ cells (PGCs) as a template. Next, for the purpose of verifying the effects of this method, cell proliferation of female primordial germ cells was examined. Female primordial germ cells cultured for about 3 months by this method were seeded at 1 × 10 ³ per well in a 24-well plate. For comparison, about 4 months have passed with female primordial germ cells after 40 days from the start of culture with centrifugation at 300 g for 5 minutes at the time of passage (frequency once or twice a week), which is the conventional method. Male primordial germ cells were seeded in the same manner, and the number of cells was counted over time. As shown in FIG. 11B, a significant increase in the number of female primordial germ cells is not recognized by the conventional method after 8 days from the start of the culture, but an approximately 4-fold increase is observed by culturing female primordial germ cells by this method. Although the rate of increase was slower than that of male primordial germ cells, this method allowed stable growth of female primordial germ cells. Stable cells for transplantation of female primordial germ cells into recipient embryos, establishment of progeny derived from transplanted cells, and gene manipulation using female primordial germ cells (stable gene transfer, knock-in, knock-out by genome editing, etc.) The increase in the number is indispensable, and stable cell culture and cell growth technology of female primordial germ cells by this method are considered to be possible for the first time.

Example 7
Establishment of cultured female primordial germ cell-derived chicken progeny After culturing female primordial germ cells for 172 days by the method described in Example 6, 2000 cells were obtained in the same manner as in Example (1-3). Transplantation was performed by microinjection into the blood of 2.5 day embryos (recipient embryos). Prior to transplantation, 6 Gy of gamma irradiation was performed. This individual (female chimera chick) was hatched, and after sexual maturation, it was crossed with a wild type Plymouth Rock species wild type. The transplanted cell-derived progeny can be distinguished from the black-haired recipient-derived progeny by the white hair of the Highline Maria species (dominant inheritance). As shown in FIG. It was shown that long-term culture with the ability to differentiate gametocytes of female primordial germ cells is possible.

Example 8
Genetic manipulation of female primordial germ cells Furthermore, it was verified whether or not long-term culture was possible by this culture method, so that stable introduction of foreign genes into female primordial germ cells was possible. The sequence consisting of the EGFP, neomycin resistance gene unit represented by SEQ ID NO: 48 was inserted into the EcoRI-EcoRV site downstream of the EF1α promoter of the vector pB530A-2 (System Bioscience, USA) having a transposon-specific terminal inverted sequence (pB530- EGFP-Neo ^r ). As described above, 0.8 μg of pB530-EGFP-Neo ^r and PiggyBac po-transposase expression vector pB200PA-1 (System Bioscience, System Bioscience, using Lipofectamine 2000 on 1 × 10 ⁵ to 5 × 10 ⁵ female primordial germ cell lines. (US) 0.8 μg of gene was simultaneously introduced. From 3 days after gene introduction, neomycin was added at a final concentration of 0.5 mg / ml and cultured. The primordial germ cells proliferating in the presence of neomycin emitted green fluorescence as shown in FIG. 12B, indicating that the foreign gene was stably introduced into the female primordial germ cells and expressed.

Furthermore, cell characteristics of female primordial germ cells into which foreign genes were stably introduced were analyzed. The female primordial germ cells emitting green fluorescence established by the above method were transplanted by microinjection into chick embryo blood 2.5 days after incubation. The number of transplanted cells per individual was 2000-5000. After the transplantation, the incubation of the embryo was continued, and the embryos on day 17 (14 days after transplantation) were analyzed. The transplanted primordial germ cells and their progeny cells that emit green fluorescence accumulated in the ovarian tissue as shown in the photograph of FIG. 12B, and were mainly localized in the cortical layer on the ventral side. Such localization is consistent with the endogenous primordial germ cells, and the female primordial germ cells have not lost their primordial germ cell characteristics through a series of culture, gene transfer, and drug selection operations. Is shown.

Example 9
Female primordial germ cell ovalbumin, ovomucoid knockout If a female primordial germ cell can be knocked out by genome editing, a female chimeric individual (G0) transplanted with the knockout female primordial germ cell can be established, and the knockout male primordial germ cell By crossing with a male chimeric individual (G0) transplanted with a homozygous knockout individual can be established in a progeny (F1 generation). This means that a knockout individual can be established one generation earlier than the conventional technology, and there are many advantages such as greatly reducing the time and cost of research. Therefore, we attempted knockout of the allergen gene using female primordial germ cells.

Using 1 × 10 ⁵ to 5 × 10 ⁵ female primordial germ cells prepared by the method described in Example 6, an attempt was made to knock out the ovalbumin gene under the same conditions as described above for male primordial germ cells It was. 1.6 μg of px330-Puro ^r -OVATg3 was transfected with Lipofectamine 2000, cultured for 2-4 days in the presence of puromycin at a final concentration of 1 μg / ml for 2 to 4 days after gene transfer, and washed for 1-2 weeks. After extracting genomic DNA from cultured cells, a partial region of the ovalbumin gene was amplified by PCR using the oligo DNA primers shown in SEQ ID NO: 21 and SEQ ID NO: 22, subcloned into a TA vector, The genomic base sequence of the region containing No. 5 (OVATg3) was analyzed. As a result, in 11 clones out of 12 clones (92%), deletion or substitution of bases was observed in the region containing SEQ ID NO: 5 (OVATg3) of the ovalbumin gene. A typical deletion is shown in FIG.

Similarly, female primordial germ cells Transgenic knockout px330-Puro ^r -OVMTg2 ovomucoid gene using, drug selection, and cultured, after extracting genomic DNA, SEQ ID NO: 23, oligo DNA shown by SEQ ID NO: 24 A partial region of the ovomucoid gene was amplified by PCR using primers, subcloned into a TA vector, and the genomic base sequence of the region containing SEQ ID NO: 9 (OVMTg2) was analyzed. Of the 12 clones analyzed, 11 clones (92%) showed a base deletion in the region containing the ovomucoid gene SEQ ID NO: 9 (OVMTg2). A typical deletion is shown in FIG. Procedures described in Example (1-3) for female primordial germ cells in which px330-Puro ^r -OVMTg2 is introduced into female primordial germ cells, drug selection, culture, and most of the ovomucoid gene is defective And transplanted into the blood of white leghorn 2.5 day embryo (recipient embryo) by microinjection. The same ionizing radiation irradiation, window opening, cell transplantation, and hatching operation as in Example (1-3) were performed, and 7 female chimeric chicks (G0) could be obtained. Based on the above, it is possible to knock out genes by genome editing by using female primordial germ cells, and establish chimeric chimeras in which female primordial germ cells accumulated in the ovaries accumulate by transplanting them into female embryos. Is possible.

Example 10
Gene knock-in to ovalbumin locus of female primordial germ cells Using female primordial germ cells, we examined whether gene knock-in was possible by genome editing like male primordial germ cells. As described above, 1 × 10 ⁵ to 5 × 10 ⁵ female primordial germ cell lines were transfected with 0.8 μg of px330-Neo ^r -OVATg1 and 0.8 μg of pBS-EGFP donor simultaneously using Lipofectamine 2000, From 3 days after introduction, puromycin was added at a final concentration of 1 μg / ml. The medium was appropriately changed, and cells proliferating in the presence of puromycin at a final concentration of 1 μg / ml were collected. After preparing genomic DNA, Nested PCR was performed to confirm knock-in of the EGFP donor construct to the ovalbumin locus ( Figure 14). Gene knock-in by genome editing is also possible in female primordial germ cells. Further, as a result of quantitative PCR similar to the test for male primordial germ cell knock-in efficiency performed in Examples 2- (2), 3- (1), 4 and 5, about 80% or more of the ovalbumin gene was found. It was judged that it was replaced by the EGFP donor construct.

Example 11
Human Primordial Germ Cell Human Interferon Gene Knock-In Using female primordial germ cells, gene knock-in of IFNβ donor construct was performed by genome editing in the same manner as male primordial germ cells performed in Example 3- (1). In the same manner as in Example 3- (1), knocking in female chick primordial germ cells and selecting with puromycin, performing Nested PCR using the selected cell genome as a template, the ovalbumin gene of the IFNβ donor construct Knock-in to the locus was confirmed (Figure 15).

Claims

A method for gene modification of poultry primordial germ cells, which comprises modifying genes of poultry primordial germ cells by genome editing.
The genetic modification method according to claim 1, wherein the poultry primordial germ cells are female primordial germ cells.
The gene modification method according to claim 1 or 2, wherein the gene is an egg protein gene.
The gene modification method according to any one of claims 1 to 3, wherein the gene is modified by genome editing using TALEN or CRISPR.
The gene modification method according to claim 4, wherein the gene is modified by genome editing using CRISPR.
The gene modification method according to any one of claims 1 to 5, wherein the gene modification is a knock-in, knock-out or partial deletion of the gene.
7. The gene according to claim 1, wherein the gene is modified by knock-in, a drug resistance gene is incorporated into the poultry primordial germ cell, and a primordial germ cell genetically modified based on the drug resistance gene is selected. 2. The gene modification method according to item 1.
The gene is modified by knockout or partial deletion, a drug resistance gene is introduced into the poultry primordial germ cell, and the primordial germ cell genetically modified based on the drug resistance gene is selected. 7. The gene modification method according to any one of 1 to 6.
Drug resistance gene is the puromycin resistance gene (Puro r) or zeocin resistance gene (Zeo r), genetically modified method according to claim 7 or 8.
The gene modification method according to any one of claims 1 to 9, wherein genome editing is performed using a plasmid vector or a viral vector.
The gene modification method according to claim 10, wherein a plasmid vector or a virus vector is introduced into an early poultry embryo, genome editing of the endogenous primordial germ cell is performed, and the endogenous poultry primordial germ cell is genetically modified.
A poultry primordial germ cell obtained by the genetic modification method according to any one of claims 1 to 10 and genetically modified by genome editing.
13. A poultry primordial germ cell according to claim 12, which is a female primordial germ cell.
A step of transplanting genetically modified poultry primordial germ cells obtained by the method of any one of claims 1 to 10 to a blastoderm, blood or gonad region of an early poultry embryo to obtain a genetically modified chimeric individual, A method for producing a genetically modified poultry, comprising the step of maturing a chimeric individual with a wild type individual, a genetically modified individual or a genetically modified chimeric individual.
A gene comprising the step of maturing a chimeric individual containing endogenous poultry primordial germ cells genetically modified by the method according to claim 11 and mating with a wild type individual, a genetically modified individual or another genetically modified chimeric individual Modified poultry production method.
The method according to claim 14 or 15, wherein the genotype of the genetically modified poultry is a mutant homo (-/-).
The method according to any one of claims 14 to 16, wherein the genetic modification is a knockout of at least one in ovo protein gene selected from the group consisting of ovalbumin, ovomucoid, ovomucin, ovotransferrin and ovoinhibitor.
The method according to any one of claims 14 to 16, wherein the genetic modification is a heterogeneous or homozygous knock-in of a foreign gene in an in ovo protein gene, and the female genetically modified poultry egg contains an expression product of the foreign gene.
18. At least one in-vitro allergen selected from the group consisting of ovalbumin, ovomucoid, ovomucin, ovotransferrin, ovoinhibitor, ovoglobulin, and lysozyme, obtained from female genetically modified poultry produced by the method of claim 17. Knockout poultry eggs with reduced or eliminated protein.
A knock-in poultry egg containing an exogenous gene expression product obtained from a female genetically modified poultry produced by the method of claim 18.
The knock-in poultry egg according to claim 20, wherein the foreign gene is a gene encoding a human-derived protein.
The foreign gene is selected from the group consisting of an antibody or a fragment thereof, an enzyme, a hormone, a growth factor, a cytokine, an interferon, a collagen, an extracellular matrix molecule, a vaccine, an agonistic protein, and an antagonistic protein. Knock-in poultry eggs.
A method for subculturing female primordial germ cells, wherein the medium is exchanged at normal pressure or under low gravitational acceleration.