Birds, methods of producing birds, and eggs of birds

Abstract

The present application provides a method for producing a bird, comprising the steps of: a modification step of cutting and modifying an ovomucin locus of a cell having pluripotency and germ cell differentiation ability of a bird using a transcription activator-like effector nuclease; and a transplantation step of transplanting the cell in which the ovomucin locus is modified into an embryo of a bird, the transcription activator-like effector nuclease being a first nuclease and a second nuclease, the first nuclease comprising the amino acid sequence shown in SEQ ID NO: 4, and the second nuclease comprising the amino acid sequence shown in SEQ ID NO: 5, in the modification step, a single vector obtained by integrating a vector expressing the first nuclease and a vector expressing the second nuclease into one vector is introduced into the cell.

Description

[0001] This application is a divisional application of the Chinese invention patent filed on July 20, 2016, with a priority date of August 27, 2015, application number “201680049500.1”, and entitled “Birds, a method for producing birds and bird eggs”. Technical Field

[0002] This invention relates to birds, methods for producing birds, and bird eggs. Background Technology

[0003] Eggs rank first among food allergies in Japan. Several proteins in eggs are known as allergens that trigger food allergies. Examples of egg allergens include ovomucoid, ovalbumin, lysozyme, and ovotransferrin.

[0004] To avoid triggering food allergies in eggs, people have tried to remove the aforementioned allergens from eggs. To remove allergens from eggs, it is simply a matter of producing chickens whose genes encoding the allergens have been disrupted.

[0005] Non-patent literature 1 discloses a genetically modified chicken that uses a transcription activator-like effector nuclease (TALEN) to knock out the ovalbumin gene. Based on this genetically modified chicken, it is believed that eggs without ovalbumin can be obtained.

[0006] Existing technical documents and patent documents

[0007] Non-patent literature 1: Park TS, 4 others, “Targeted gene knockout in chickens mediated by TALENs.”, Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(35), 12716-12721. Summary of the Invention

[0008] Technical issues

[0009] Even eggs that do not contain ovalbumin contain approximately 10% ovomucoid in their egg white. Ovomucin is the most allergenic protein in eggs. Due to its high physicochemical stability, ovomucoid maintains its allergenicity even when heated. Therefore, according to the method disclosed in Non-Patent Literature 1, even if ovalbumin is removed from the egg or ovalbumin is inactivated by heating, it is difficult to say that the allergenicity of the egg will be sufficiently reduced.

[0010] Furthermore, non-patent document 1 mentioned above uses primordial germ cells. Since the number of primordial germ cells present during their production is very small, a technique for culturing primordial germ cells is necessary. To date, several methods for culturing primordial germ cells have been reported, but these reports are limited to a few research institutions. Even when culturing according to the reported methods, primordial germ cells cannot be cultured, thus presenting a lack of certainty.

[0011] In view of the above facts, the present invention aims to provide a bird that can sufficiently reduce the allergenicity of its eggs, a method for producing birds, and bird eggs with sufficiently reduced allergenicity.

[0012] Solution to the problem

[0013] The inventors knocked out the ovomucoid gene in chickens using existing homologous recombination methods. However, in existing homologous recombination methods, cell culture requires a long time, resulting in cell damage and visible abnormal karyotypes. Moreover, even when chimeric chickens obtained through homologous recombination are mated together, it is impossible to obtain homologous conjugate chickens with the ovomucoid gene knocked out. The eggs laid by these chimeric chickens contain ovomucoid from the recipient genome, and the allergenicity of the eggs is not sufficiently reduced.

[0014] Therefore, the inventors conducted repeated and in-depth research and completed this invention by applying genome editing technology. That is:

[0015] The first aspect of the present invention relates to birds whose eggs do not contain artificially introduced foreign genes in their genome, and whose egg mucin content is lower than that of wild types.

[0016] In this case, the birds described in the first aspect of the present invention may contain a stop codon in at least one of the first to third exons, counting from the 5' end of the oocyte mucin locus, or

[0017] Starting from the 5' end of its ovomucoid locus, the first exon may not contain a start codon.

[0018] Furthermore, in the birds described in the first aspect of the present invention, the signal sequence in the oocyte mucin locus can be modified.

[0019] In addition, the birds involved in the first aspect of the present invention described above can be chickens.

[0020] The second aspect of the present invention relates to a method for producing birds, which includes the following steps:

[0021] The modification step involves cutting and modifying the oval mucin locus of avian pluripotent stem cells using a programmable endonuclease; and

[0022] The transplantation step involves transplanting the aforementioned pluripotent stem cells, modified with the oval mucin gene locus, into the embryo of a bird.

[0023] In this case, the aforementioned programmable endonuclease can be a transcription activator-like effector nuclease.

[0024] Furthermore, the aforementioned transcription activator-like effector nucleases can be either the first nuclease or the second nuclease.

[0025] The first nuclease mentioned above may contain the amino acid sequence shown in sequence number 4.

[0026] The second nuclease mentioned above may contain the amino acid sequence shown in sequence number 5.

[0027] Furthermore, the aforementioned transcription activator effector nucleases can be either 3rd or 4th nucleases.

[0028] The aforementioned third nuclease may contain the amino acid sequence shown in sequence number 6.

[0029] The aforementioned fourth nuclease may contain the amino acid sequence shown in sequence number 7.

[0030] In addition, the programmable endonuclease mentioned above can be the Cas9 nuclease in the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) associated protein system.

[0031] In addition, the Cas9 nuclease described above can cleave double-stranded DNA in the region of the oval mucin locus containing the nucleotide sequence shown in sequence number 8.

[0032] In addition, the Cas9 nuclease described above can cleave double-stranded DNA in the region of the oval mucin locus containing the nucleotide sequence shown in sequence number 9.

[0033] In addition, the Cas9 nuclease described above can cleave double-stranded DNA in the region of the oval mucin locus containing the nucleotide sequence shown in sequence number 10.

[0034] In addition, the Cas9 nuclease described above can cleave double-stranded DNA in the region of the oval mucin locus containing the nucleotide sequence shown in sequence number 11.

[0035] In addition, the Cas9 nuclease described above can cleave double-stranded DNA in the region of the oval mucin locus containing the nucleotide sequence shown in sequence number 12.

[0036] In addition, the Cas9 nuclease described above can cleave double-stranded DNA in the region of the oval mucin locus containing the nucleotide sequence shown in sequence number 13.

[0037] In addition, the above modification steps can modify the oval mucin locus from pluripotent stem cells from the epiblast.

[0038] The bird eggs involved in the third aspect of the present invention do not contain artificially introduced foreign genes in their genome, and...

[0039] The content of oval mucin is lower than that of wild type.

[0040] Invention Effects

[0041] According to the present invention, the allergenicity of eggs can be sufficiently reduced. Furthermore, according to the present invention, bird eggs with sufficiently reduced allergenicity can be obtained. Attached Figure Description

[0042] Figure 1 This is a diagram showing the nucleotide sequences of the first exon (exon 1), the second exon (exon 2), and the third exon (exon 3) from the 5' end in the chicken oocyte mucin locus.

[0043] Figure 2 This is a diagram showing the TALEN recognition regions identified in exon 1 and exon 3.

[0044] Figure 3 This is a graph showing the relative cleavage activity of TALENs targeting exon 1 and exon 3.

[0045] Figure 4 This is a diagram showing the nucleotide sequence of the target region in the genome of pluripotent stem cells from the epiblast that have been implanted with a TALEN expression vector targeting exon 3.

[0046] Figure 5This is a graph showing the relative cleavage activity of TALENs targeting exon 3 and highly active TALENs targeting exon 1.

[0047] Figure 6 This is a diagram showing the composition of the TALEN expression vector.

[0048] Figure 7 This is a graph showing the relative cleavage activity of TALEN in chicken cells.

[0049] Figure 8 This diagram shows the introduction of mutations into pluripotent stem cells derived from the epiblast via the TALEN expression vector. (A) shows the results of genomic PCR (polymerase chain reaction). (B) shows the results of Cel-I assay.

[0050] Figure 9 This is a diagram showing a portion of the nucleotide sequence of the cloned oval mucin locus and the amino acid sequence encoded by that nucleotide sequence. (A), (B), and (C) show clones with knockout mutations. (D) shows the wild type.

[0051] Figure 10 This is a diagram showing colonies of cloned pluripotent stem cell lines from the epiblast with knockout mutations.

[0052] Figure 11 This is a diagram showing a portion of the nucleotide sequence of the oocyte mucin locus in cloned pluripotent stem cell lines with knockout mutations from the epiblast and in wild-type cells.

[0053] Figure 12 This is a diagram showing a portion of the nucleotide sequence of the ovomucoid locus from a cloned pluripotent stem cell line with a knockout mutation, along with the amino acid sequence encoded by that nucleotide sequence and a portion of the amino acid sequence encoded by the wild-type ovomucoid locus. (A) Shows the nucleotide and amino acid sequences of pluripotent stem cell line #4 from the epiblast. (B) Shows the nucleotide and amino acid sequences of pluripotent stem cell lines #5 and #5-3 from the epiblast.

[0054] Figure 13 These are images showing photographs of knockout chimeric chickens produced from pluripotent stem cell lines with knockout mutations derived from the epiblast. (A) shows a chimeric chicken from pluripotent stem cell line #5, which is derived from the epiblast. (B) shows a chimeric chicken from pluripotent stem cell line #4, which is derived from the epiblast.

[0055] Figure 14 This is a diagram showing the composition of the CRISPR / Cas9 vector used for ovomucoid knockout.

[0056] Figure 15 This is a diagram showing the nucleotide sequences of the oligoDNAs inserted into the CRISPR / Cas9 vector for ovomucoid knockout.

[0057] Figure 16 This is a graph showing the relative cleavage activity of the CRISPR / Cas9 vector for ovomucoid knockout in HEK293 cells.

[0058] Figure 17 This is a graph showing the relative cleavage activity of ovomucoid knockout using the CRISPR / Cas9 vector in pluripotent stem cells derived from the epiblast. Detailed Implementation

[0059] Embodiments of the present invention will be described. However, the present invention is not limited to the following embodiments and drawings.

[0060] (Implementation Method 1)

[0061] First, Implementation Method 1 will be described. The eggs laid by the birds involved in Implementation Method 1 do not contain artificially introduced foreign genes in their genome, and the content of oocyte mucin is lower than that of the wild type.

[0062] There are no specific restrictions on the types of birds; examples include chickens, ducks, turkeys, wild ducks, geese, quails, pheasants, parrots, sparrows, eagles, ostriches, emus, and cassowaries. Chickens are preferred. There are no specific restrictions on the breeds of chickens; examples include White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Iihode Island, Australorp, Minorca, Amrox, California Gray, Italian Partidge, and Korean Oge.

[0063] The genome of the birds involved in this embodiment does not contain any artificially introduced foreign genes. Here, "artificially introduced foreign genes" refers to mutated genes introduced artificially through gene recombination technology or other means, and genes that are not originally present in the bird's genome. Methods for artificially introducing foreign genes into the genome include homologous recombination, retroviral vector methods, lentiviral vector methods, and artificial viral vector methods. The genome of the aforementioned birds does not contain foreign genes introduced through these methods.

[0064] Ovomucin is a heat-resistant glycoprotein with a molecular weight of approximately 28,000. It is produced by secretory cells of the oviduct. Ovomucin is typically found primarily in the albumen of bird eggs. For example, it accounts for approximately 11% by weight of the albumen in an egg. The ovomucin content in the eggs laid by the birds of this embodiment is lower than that in wild-type eggs of the same species. Here, wild-type refers to birds of the same species as described above that have not undergone genetic modification.

[0065] The content of oval mucin in eggs can be quantified using known methods for detecting target proteins. For example, using samples prepared from egg whites collected from wild-type eggs and eggs laid by birds of this embodiment, in Western blotting, the content of oval mucin can be compared based on the color intensity of the bands by immunostaining with antibodies that bind to oval mucin.

[0066] To ensure quantification, the content of ovomucoid is preferably determined by a sandwich ELISA (Enzyme-Linked ImmunoSorbent Assay). The capture and detection antibodies used in the sandwich ELISA can be monoclonal or polyclonal antibodies. Examples of capture and detection antibodies include rabbit anti-ovomucoid antibody and mouse anti-ovomucoid antibody, respectively.

[0067] As a rabbit anti-ovomucoid antibody, any polyclonal antibody obtained by recovering antiserum from rabbits immunized with ovomucoid and then purifying it using affinity chromatography of ovomucoid is acceptable. Conversely, as a mouse anti-ovomucoid antibody, any mouse anti-ovomucoid antibody obtained by generating a hybridoma using a monoclonal antibody against ovomucoid through cell fusion of mouse spleen cells and then purifying it from ascites antibody is acceptable. There are no particular limitations on the detection antibody; peroxidase labeling is sufficient. When labeled with peroxidase, ovomucoid can be quantified based on the color development of TMB (3,3',5,5'-tetramethylbenzidine). The content of ovomucoid can be evaluated based on its concentration in the sample. By constructing an appropriate sandwich ELISA, ovomucoid can be quantified at a detection limit of approximately 50 pg / ml.

[0068] By quantifying the ovalbumin content in the manner described above, it can be confirmed that the ovalbumin content in the eggs laid by the birds involved in this embodiment is lower than that in wild-type birds of the same species. For example, the ovalbumin content in the eggs laid by the birds involved in this embodiment is 95% or less by weight, 80% or less by weight, 60% or less by weight, 40% or less by weight, 20% or less by weight, 10% or less by weight, or 5% or less by weight compared to the ovalbumin content in wild-type eggs of the same species. Furthermore, when quantifying the ovalbumin in the eggs laid by the birds involved in this embodiment, the concentration of ovalbumin can be below the detection limit. In particular, when the birds involved in this embodiment are chickens, the ovalbumin content in the ovalbumin of the eggs laid by these chickens can be 0-8% by weight, 0-4% by weight, 0-3% by weight, 0-2% by weight, or 0-1% by weight. In addition, the ovalbumin content can be evaluated based on the weight of ovalbumin per unit volume of ovalbumin.

[0069] It is particularly preferred that the eggs laid by the birds involved in this embodiment do not contain ovomucin. Furthermore, "without ovomucin" means that the ovomucin contained in the egg white of wild-type eggs is absent. Therefore, eggs without ovomucin also include eggs containing non-full-length ovomucin fragments.

[0070] In the ovomucoid locus of the avian genome involved in this embodiment, there exists a knockout mutation that prevents ovomucoid expression or a mutation that prevents the full-length ovomucoid from being expressed, thus ovomucoid is not normally expressed. This mutation is arbitrary, as long as ovomucoid is not normally expressed; specifically, it is preferable to insert a stop codon into an exon within the ovomucoid locus.

[0071] The ovomucoid locus contains five exons. When counting exons 1 through 5 from the 5' end, the stop codon can be inserted at any position in exons 1 through 5. More preferably, the aforementioned birds contain a stop codon in at least one of exons 1 through 3 at the ovomucoid locus. When a stop codon is inserted in exons 1 through 5 within the ovomucoid locus, ovomucoid is not fully synthesized, and the full-length ovomucoid is not expressed. Depending on the position of the stop codon insertion, although a fragment of ovomucoid is expressed, the epitope of the ovomucoid fragment is reduced compared to the full-length ovomucoid, thus lowering allergenicity.

[0072] Alternatively, the mutation could be a mutation in the start codon within the ovomucoid locus. When a mutation exists in the start codon within the ovomucoid locus, ovomucoid synthesis can proceed solely based on the mRNA. For example, the aforementioned birds do not have a start codon in exon 1 of the ovomucoid locus.

[0073] Preferably, the signal sequence in the oval mucin locus of the aforementioned birds is modified. The signal sequence in the oval mucin locus contains a portion of the nucleotide sequence of exon 1 and a portion of the nucleotide sequence of exon 2, encoding 25 amino acid residues. Figure 1 The nucleotide sequences of exon 1, exon 2, and exon 3 are displayed. Figure 1 In the text, the bases of exons are displayed in large letters, while the bases of introns are displayed in small letters. Figure 1 The underlined nucleotide sequence shown is the signal sequence. Not only is the full-length ovomucoid not expressed, but even fragments of ovomucoid are not expressed. In modifying the signal sequence, mutations can be introduced into the ATG (start codon) or into the signal sequence to produce a stop codon. Preferably, the stop codon is contained in exon 1 of the ovomucoid locus. Furthermore, the nucleotide sequences of exon 1, exon 2, and exon 3 are shown as in sequence numbers 1, 2, and 3, respectively.

[0074] As stated above, the aforementioned birds do not contain artificially introduced foreign genes in their genomes. Therefore, in the production of these birds with modified oval mucin loci, homologous recombination, which replaces foreign genes with specific genes in the genome, is not used; instead, genome editing techniques detailed below are preferred.

[0075] As detailed above, the birds involved in this embodiment lay eggs with lower levels of oocyte mucin compared to the wild type because oocyte mucin is not normally expressed. In oocyte allergy testing, even when oocyte mucin is examined independently (excluding yolk and albumen), its allergenicity is high. Therefore, eggs with lower oocyte mucin content than the wild type can sufficiently reduce oocyte allergenicity.

[0076] Furthermore, the aforementioned birds do not contain any artificially introduced foreign genes in their genomes. The absence of foreign genes prevents the emergence of unintended phenotypes and toxicities. Additionally, the absence of foreign genes minimizes the possibility of compromising the certainty of reproductive inheritance in these birds.

[0077] Furthermore, due to the high physicochemical stability of ovomucoid, its allergenicity can be maintained even in processed foods or vaccines containing avian egg whites that have undergone heat treatment. Therefore, eggs with a lower ovomucoid content than wild-type eggs, as described in this embodiment, are also useful as raw materials for various products such as processed foods and vaccines.

[0078] Furthermore, the aforementioned birds may contain a stop codon in at least one exon of exons 1-3 of their oval mucin locus. When exon 3 contains a stop codon, the antigenic determinant of the secreted oval mucin fragment is less than that of the full-length oval mucin, thus reducing allergenicity. In particular, since exon 1 of the oval mucin locus contains a stop codon, the birds according to this embodiment can lay eggs that contain not only no full-length oval mucin but also no oval mucin fragments at all. In addition, when exon 1 of the oval mucin locus does not contain a start codon, oval mucin cannot be synthesized based on mRNA, so the aforementioned birds can lay eggs that do not contain oval mucin.

[0079] Furthermore, the signal sequence at the oval mucin locus in these birds can be modified. Since the signal peptide corresponding to the signal sequence is cleaved within the endoplasmic reticulum and not secreted, the presence of a stop codon in the signal sequence can prevent the peptide from the oval mucin locus from being secreted into the egg white. As a result, the allergenicity caused by oval mucin can be further reduced.

[0080] Furthermore, the preferred bird species in this embodiment is the chicken. Since chickens lay eggs in high demand, this allows for an efficient supply of eggs with significantly reduced allergenicity. In addition to chickens, quail eggs are also preferred for supplying eggs intended for consumption.

[0081] In addition, mutations in the ovomucoid locus can be mutations that cause frameshifting into the exons within the ovomucoid locus.

[0082] In another embodiment, a bird egg is provided that does not contain artificially introduced foreign genes in its genome and has a lower content of ovalbumin than that of wild-type eggs. In particular, eggs are widely used as a raw material for snacks, beverages, processed foods, etc., or for manufacturing pharmaceuticals such as vaccines. When using the egg according to this embodiment, even if ovalbumin is mixed into various products, the allergenicity of ovalbumin can be reduced. Moreover, when using bird eggs that do not contain ovalbumin, the risk of the most allergenic ovalbumin being mixed into various products can be greatly suppressed.

[0083] (Implementation Method 2)

[0084] Next, Embodiment 2 will be described. In Embodiment 2, a method for producing birds suitable for the birds described in Embodiment 1 will be explained.

[0085] When producing birds that lay eggs with lower levels of ovomucin than the wild type, it is necessary to produce birds whose ovomucin loci have been modified. To produce genetically modified animals, the genome of a one-cell stage fertilized egg must be modified, and the resulting individuals are then screened for the introduction of the target mutation. Therefore, in the production of genetically modified animals, fertilized eggs need to be manipulated in vitro to produce individuals. For example, in mice and rats, multiple unfertilized eggs can be obtained from a single female. Therefore, after in vitro fertilization, fertilized eggs are produced up to the one-cell stage, the genome is modified, and the fertilized eggs are returned to the ovary of the female, thus allowing for the creation of multiple individuals.

[0086] On the other hand, in vitro fertilization technology for birds has not yet been established. Furthermore, only one one-cell stage fertilized egg can be obtained from a single egg-laying bird. Moreover, it is difficult to determine the specific one-cell stage fertilized egg present in the oviduct. For these reasons, it is difficult to apply gene modification technology to bird fertilized eggs. Therefore, in order to produce genetically modified birds, pluripotent stem cells with pluripotency or germ cell differentiation capacity are used instead of fertilized eggs.

[0087] Therefore, the bird production method according to this embodiment includes the following steps: a modification step, in which the oval mucin locus of bird pluripotent stem cells is cut and modified with a programmable endonuclease; and a transplantation step, in which the above-mentioned pluripotent stem cells modified with the oval mucin locus are transplanted into the embryo of a bird.

[0088] First, the above modification steps will be explained in detail. As pluripotent stem cells for birds, embryonic stem cells (ES cells) possessing pluripotency and germ cell differentiation capacity can be cited as examples. Bird ES cells are, for example, epiblast-derived stem cells (hereinafter referred to simply as "epiSC"), which can be established from blastodiscal cells isolated from the epiblast of a fertilized egg. In the case of chickens, the blastodisc of stage X in the Eyal-Giladi and Kochav generation stages (I-XIV) contains the epiblast. Chicken epiSCs are obtained, for example, by culturing blastodiscal cells isolated from the epiblast on supporting cells using known methods, wherein the supporting cells have had their cell proliferation stopped by irradiation or treatment with mitomycin C. Examples of supporting cells include chicken embryonic fibroblasts, mouse embryonic fibroblasts, and cell lines derived from mouse embryonic fibroblasts.

[0089] Furthermore, primordial germ cells can be utilized to possess pluripotency and germ cell differentiation capabilities. Primordial germ cells can, for example, be isolated from the gonads of avian fetuses using known methods. Additionally, the use of epiSC in the modification steps will be explained below.

[0090] In the modification of ovomucoid loci, programmable endonucleases are used to cut ovomucoid loci on the genome, such as epiSC, at specific sites. Programmable endonucleases are used in so-called genome editing technologies that can specifically modify (delete, substitute, insert) target sites on the genome. Programmable endonucleases are designed based on the nucleotide sequence of the target DNA and can cut DNA with any nucleotide sequence. There are no particular limitations on programmable endonucleases; examples include TALEN, zinc finger nucleases (ZFNs), and Cas9 nucleases in the CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat and Crisper associated protein) system (also known as "CRIPPR / Cas9").

[0091] TALEN and ZFN are polypeptides containing DNA-binding and DNA-cleaving domains. At the binding site of the DNA-binding domain, a pair of DNA-cleaving domains of TALEN and ZFN approach each other to form a dimer, thereby cleaving double-stranded DNA. The DNA-binding domain repeatedly contains multiple DNA-binding modules, each recognizing a specific base pair in the DNA. Therefore, by appropriately designing these DNA-binding modules, it is possible to specifically cleave the nucleotide sequence at the ovomucoid locus as a target.

[0092] The CRISPR-Cas system uses guide RNA and the Cas9 nuclease. The guide RNA has a nucleotide sequence adjacent to the PAM sequence on the genome that is complementary to the target nucleotide sequence. The guide RNA comprises CRISPR RNA (crRNA) complementary to the target nucleotide sequence and an auxiliary tracrRNA. The Cas9 nuclease cleaves double-stranded DNA in the region formed by the target nucleotide sequence at the 5' end of the PAM sequence. The Cas9 nuclease recognizes the guide RNA that binds to the target nucleotide sequence at the ovomucoid locus.

[0093] The cutting of double-stranded DNA using programmable endonucleases is a cause of loss of multiple genetic information or cancer, and is therefore repaired very rapidly within cells. During non-identical end-binding repair, which is one of the main repair pathways and involves the joining of the cut ends, mutations (deletions or insertions) are introduced into the nucleotide sequence of the genome with a high probability. Therefore, when using TALENs, TALENs exist at the 5' and 3' ends of the sites to be modified at the ovomucoid locus, and TALENs can be designed based on the nucleotide sequences (effect sequences) of the regions recognized by the DNA-binding module. Effector sequences suitable for the sites to be modified at the ovomucoid locus can be specified, for example, through "TALEN Targeter" (https: / / tale-nt.cac.cornell.edu / ). Alternatively, when using the CRISPR-Cas system, target nucleotide sequences in the ovomucoid gene can be specified, for example, through "CRISPR direct" (http: / / crispr.dbcls.jp / ).

[0094] In the modification step, vectors expressing programmable endonucleases can be introduced into epiSC using known methods such as microinjection, electroporation, calcium phosphate transfection, and lipid transfection. For example, when introducing a vector expressing TALEN, a programmable endonuclease, into epiSC, a TALEN expression vector designed based on the target nucleotide sequences of each double strand of genomic DNA is used. When using the CRISPR-Cas system as a programmable endonuclease, vectors expressing guide RNA and Cas9 nuclease can be similarly introduced into epiSC.

[0095] In modifying the ovomucoid locus, a mutation can be introduced into any site of the ovomucoid locus, preferably into at least one exon of exons 1-3 or the signal sequence, in a manner that does not express ovomucoid. Preferably, the mutation causes at least one exon of exons 1-3 to contain a stop codon, or causes exon 1 to omit a start codon. Therefore, a programmable endonuclease can be designed to cleave at least one exon of exons 1-3 or the signal sequence. The programmable endonuclease is designed according to known methods based on the nucleotide sequence near the cleavage site.

[0096] Specifically, TALEN has TALEN left (first nuclease) and TALEN right (second nuclease). When cleaving exon 1, for example, TALEN left and TALEN right contain the amino acid sequences shown in sequence numbers 4 and 5, respectively. Furthermore, when cleaving exon 3, for example, TALEN left (third nuclease) and TALEN right (fourth nuclease) contain the amino acid sequences shown in sequence numbers 6 and 7, respectively.

[0097] Furthermore, when TALEN left has nuclease activity that specifically cleaves the target nucleotide sequence, it can include amino acid sequences obtained by deleting, substituting, or adding one or more amino acids in the amino acid sequences shown in sequence number 4 or 6. When TALEN right also has nuclease activity that specifically cleaves the target nucleotide sequence, it can also include amino acid sequences obtained by deleting, substituting, or adding one or more amino acids in the amino acid sequences shown in sequence number 5 or 7.

[0098] On the other hand, in the CRISPR-Cas system, for example, when cleaving exon 1, the Cas9 nuclease only needs to cleave the double-stranded DNA in the region containing any of the nucleotide sequences shown in sequence numbers 8-11 of the ovomucoid locus. Similarly, when cleaving exon 2, the Cas9 nuclease only needs to cleave the double-stranded DNA in the region of the ovomucoid locus containing the nucleotide sequences shown in sequence number 12 or 13.

[0099] To introduce the guide RNA into epiSC, an oligoDNA expressing the guide RNA is preferably used. The nucleotide sequence of the oligoDNA is determined based on the target nucleotide sequence. When the Cas9 nuclease cleaves the region containing the nucleotide sequence shown in SEQ ID NO: 8, the sense nucleotide sequence of the oligoDNA expressing the guide RNA in epiSC contains the nucleotide sequence shown in SEQ ID NO: 14, while the antisense nucleotide sequence of the oligoDNA contains the nucleotide sequence shown in SEQ ID NO: 15. Furthermore, when the nucleotide sequence of the region cleaved by the Cas9 nuclease is any one of the nucleotide sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, the combinations of nucleotide sequences contained in the sense and antisense nucleotide sequences of the oligoDNA can be listed as SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, and SEQ ID NO: 20 and SEQ ID NO: 21, respectively. In addition, when the nucleotide sequence of the region cleaved by the Cas9 nuclease is any of the nucleotide sequences shown in sequence number 12 and sequence number 13, the combinations of nucleotide sequences contained in the sense and antisense nucleotide sequences of the above-mentioned oligoDNA can be listed as sequence number 22 and sequence number 23, and sequence number 24 and sequence number 25, respectively.

[0100] Whether the ovomucoid locus has been modified in the above modification steps can be determined by analyzing the nucleotide sequence of the ovomucoid locus on the epiSC genome. For example, after introducing a programmable endonuclease, the genomic DNA can be recovered from the stably proliferating epiSC, and then the nucleotide sequence of the ovomucoid locus can be analyzed.

[0101] Furthermore, to efficiently obtain chimeric individuals with genomes modified in a manner that does not express ovomucoid, epiSCs can be screened for cells containing a knockout mutation in which the ovomucoid gene is not expressed. Additionally, to concentrate cells containing vectors expressing programmable endonucleases, expression systems that transiently express drug resistance genes such as puromycin can be introduced into epiSCs.

[0102] Next, the transplantation procedure will be described in detail. In this procedure, epiSC, modified with the oocyte mucin locus, is transferred into the avian embryo. There are no particular limitations on the transplantation technique; simply injecting epiSC into the avian embryo using a thin tube is sufficient.

[0103] Specifically, in the transplantation step, for example, epiSC, which modifies the ovomucin locus in a manner that does not express ovomucin, can be transplanted into the blastodisc of a newly laid fertilized egg embryo that has been irradiated with gamma rays. At this time, chimeric individuals can be easily identified based on feather color by using a receptor from a system with a different feather color than the epiSC system. For example, in producing chimeric chickens, it is preferable to transplant epiSC from the barred Plymouth Rock breed (black feathers) into the embryo of a white-feathered Lorikeet breed (white feathers).

[0104] Following the transplantation step, chimeric individuals can be produced by incubating eggs containing embryos transplanted with epiSC. In chickens, the incubation period is approximately 20 days. Within the chimeric individual, sperm and eggs with a genome containing a modified ovomucin locus are formed. Therefore, by mating chimeric individuals, genetically modified birds are produced with a high probability, inheriting a genome containing the modified ovomucin locus through homologous mating. Because the ovomucin locus in these genetically modified birds is modified, the ovomucin content in the eggs produced by these birds is lower than in wild-type birds, or the eggs produced by these birds may not contain ovomucin at all.

[0105] Furthermore, as mentioned above, genetically modified birds can be identified by their feather color. Additionally, genetically modified birds can also be identified by analyzing the nucleotide sequence of their genomic DNA using methods such as DNA imprinting.

[0106] As detailed above, in the bird production method of this embodiment, the oval mucin locus of avian pluripotent stem cells is cut and modified using a programmable endonuclease, thereby obtaining birds that do not normally express oval mucin. Since the genome of this bird is reproductively inherited, the oval mucin content in the eggs laid by this bird is lower than that of the wild type, or the eggs laid by this bird do not contain oval mucin at all. Therefore, the allergenicity of the eggs can be significantly reduced.

[0107] Furthermore, in the modification step of this embodiment, the ovomucoid locus of epiSC can be modified. epiSC can be easily established from approximately 60,000 blastodiscal cells obtained from one embryo and can be stably cultured while maintaining pluripotency, thus allowing for more definitive modification of the ovomucoid locus.

[0108] Example

[0109] The present invention is further illustrated by the following embodiments, but the present invention is not limited to the embodiments.

[0110] Example 1: Preparation of TALEN expression vector

[0111] (Selection of target sequences for ovomucoid)

[0112] To introduce mutations into the ovomucoid gene via TALEN, effector sequences were retrieved using the TALEN Targeter, targeting the nucleotide sequences of exons 1, 2, and 3 of the ovomucoid gene. The results are as follows: Figure 2 As shown in the underlined region, one set of effector sequences was observed in the nucleotide sequences of exon 1 and exon 3, respectively. Furthermore, no effector sequence was observed in exon 2.

[0113] (Preparation of TALEN expression vectors and study of TALEN cleavage activity)

[0114] First, modules capable of binding to the effector sequences in exons 1 and 3 were constructed using a 6-module assembly method. Two types of G-TALEN expression vectors (left and right) were fabricated in exons 1 and 3, respectively. The G-TALEN expression vectors (hereinafter referred to as "G-TALEN expression vectors") were fabricated using the G-TALEN and TAL effector kit 2.0 and the Yamamoto Lab TALEN Accessory Pack (both available from Addgene) according to the operating instructions provided with the kits.

[0115] Next, to investigate the TALEN cleavage activity, a single-strand annealing (SSA) assay was performed using HEK293 cells. In the SSA assay, as described below, a reporter vector containing the nucleotide sequence that serves as the target of TALEN and a G-TALEN expression vector were introduced together into HEK293 cells, and the cleavage activity was determined by reporter activity assay.

[0116] A reporter vector was created by inserting annealed synthetic oligoDNA into pGL4-SSA contained in the Yamamoto Lab TALEN Accessory Pack. First, pGL4-SSA was treated with BsaI, followed by electrophoresis without dephosphorylation, and then cleaved. For exon 1, the nucleotide sequences of the sense and antisense oligoDNA contained in the inserted synthetic oligoDNA are shown in SEQ ID NO. 26 and SEQ ID NO. 27, respectively. For exon 3, the nucleotide sequences of the sense and antisense oligoDNA contained in the inserted synthetic oligoDNA are shown in SEQ ID NO. 28 and SEQ ID NO. 29, respectively.

[0117] The details of the annealing solution for synthesizing oligoDNA are shown below.

[0118] 1 μl of 10× buffer (400 mM Tris-HCl (pH 8), 200 mM MgCl2, 500 mM NaCl)

[0119] Justice oligoDNA (50μM) 1μM

[0120] Antisense oligoDNA (50μM) 1μM

[0121] 7μM sterile distilled water

[0122] The annealed solution was maintained at 95°C for 5 minutes, and then cooled to 25°C for 90 minutes to anneal the synthesized oligoDNA.

[0123] Next, the annealed synthetic oligoDNA was inserted into pGL4-SSA cells treated with BsaI. The subcloned product was then cultured in a small culture medium. Treatment with KpnI revealed two bands at 3800 bp and 1800 bp, confirming the insertion of the synthetic oligoDNA.

[0124] Next, the reporter vector sequence analysis was performed in the following order. After NarI treatment, the reporter vector was subjected to electrophoresis, the gel was excised, and the gel fragments were recovered into microtubes. The microtubes were placed in a deep freezer for approximately 10 minutes to allow them to freeze completely, then thawed by warming with a finger and centrifuged. Approximately 6–8 μl of the exudate was collected and used as a template for sequencing.

[0125] Primers used for sequence analysis were Luc2-up-F (Sequence No. 30) or Luc2-down-R (Sequence No. 31). After confirming the correct nucleotide sequence was inserted, the reporter vector was purified using a transfection-grade Miniprep kit, and the concentration was quantified to 150 ng / μl.

[0126] DNA solutions containing the following four plasmids were transfected into HEK293 cells.

[0127] G-TALEN expression vector (left) 200ng

[0128] G-TALEN expression vector (right) 200 ng

[0129] Report carrier 100ng

[0130] pRL-CMV (reference vector) 20 ng

[0131] HEK293 cells were cultured in 70-80% confluence using 10 cm diameter culture dishes. The required amounts of serum-free Dulbecco's modified Eagle's medium (DMEM) diluted with DNA and serum-free DMEM diluted with Lipofectamine LTX were injected into microtubes. 25 μl of serum-free DMEM diluted with DNA was added to each well of a 96-well plate, followed by 4-8 μl of the aforementioned DNA solution, and the mixture was stirred. LTX was added to the serum-free DMEM diluted with LTX to a final concentration of 0.7 μl / well (25 μl), and the cells were resuspended. Immediately after resuspending, 25 μl of LTX was added to each well. This process was repeated for the required number of cells. The culture medium was removed from the culture dishes, and 15% fetal bovine serum (FBS) / DMEM was added. Cells were directly pipetted onto the culture dishes to resuspend the cells. Cell counts were determined using a hemocytometer, totaling 6 × 10⁶ cells / well. 5 Cells / ml.

[0132] LTX was added to the initial wells, and after 30 minutes, 100 μl of the prepared cells were added to each well. The cells were then incubated in a CO2 incubator at 37°C. Luciferase activity was measured 24 hours after transfection using the Dual-Glo Luciferase Assay System (Promega) according to the instruction manual.

[0133] (result)

[0134] Figure 3The cleavage activity of the G-TALEN expression vector obtained by SSA assay is shown. The positive control TALEN is a TALEN expression vector with sufficient cleavage activity, prepared targeting HPRT1. Relative activity refers to the relative value when the HPRT1 cleavage activity is measured using HEK293 cells, with that measurement value as 1. The negative control shows the relative activity value when the G-TALEN expression vector is introduced into HEK293 cells without the target sequence. Figure 3 As shown, no cleavage activity was detected in G-TALEN expression vectors targeting exon 1, but cleavage activity was only detected in G-TALEN expression vectors targeting exon 3.

[0135] Example 2: Introduction of mutations into chicken epiSCs using G-TALEN expression vector and confirmation of mutation introduction by CE1-I assay.

[0136] When introducing mutations into chicken epiSC, a G-TALEN expression vector targeting exon 3 with confirmed cleavage activity was used.

[0137] (Cultivation of chicken epiSC)

[0138] First, separate the germinal disc cells from freshly laid fertilized eggs in the following order. After completely removing the albumen using an egg white separator, place the fertilized eggs in a plastic dish with the germinal disc positioned above the yolk. Attach a sterilized, dried filter paper ring (made by cutting a 5mm diameter hole in filter paper and then scissors to create a circle along its outer edge) to the fertilized egg, centering the germinal disc. Insert scissors (small, straight, pointed scissors) along the outer edge of the filter paper ring and cut the germinal disc on each yolk membrane into a circle. Next, using small forceps, slowly tilt and lift the filter paper to remove as much yolk as possible from the filter paper ring. At this point, the epiblast is adhered to the filter paper ring.

[0139] Next, with the yolk side facing up, immerse the filter paper ring in a dish containing sterile PBS (phosphate-buffered saline) and gently shake the ring to remove any attached yolk. Transfer the filter paper ring to another prepared dish containing sterile PBS and shake vigorously to separate the blastodiscal cells from the ring into a disc shape. Collect the separated blastodiscal cells into a 1.5 ml tube using a micropipette.

[0140] Next, the isolated embryonic disc cells were cultured on pre-prepared supporting cells. A cell line derived from mouse embryonic fibroblasts (STO cells) was used as the supporting cells. The preparation of the supporting cells is described below.

[0141] STO cells were seeded in 10 cm diameter culture dishes. The culture medium was 10% FBS (fetal bovine serum)-DMEM (Dulbecco's modified Eagle medium). Culture was performed at 37°C with 5% CO2. After approximately 3 days of culture, the STO cells reached confluence. They were washed three times with cold PBS, then peeled with 0.025% trypsin and 0.02% EDTA 2Na-PBS, and seeded in 15 cm diameter culture dishes. Once confluence was achieved, mitomycin C was added to the culture medium to a final concentration of 10 μg / ml, and the cells were cultured for 2 hours. They were washed five times with cold PBS, peeled with 0.025% trypsin and 0.02% EDTA 2Na-PBS, and centrifuged at least three times. The cell count was determined using a hemocytometer.

[0142] For the culture of Support cells, gelatin coating was used in culture dishes with a diameter of 6 cm. The gelatin coating solution was added to distilled water to achieve a gelatin content of 0.1% before use, followed by dissolution and sterilization using an autoclave. At least 2 hours before culturing the Support cells, the culture dishes were immersed in the gelatin coating solution at 37°C. After removing the gelatin coating solution, the mitomycin C-treated Support cells were adjusted and seeded in 10% FBS-DMEM, with each 6 cm diameter culture dish containing 2–3 × 10⁶ cells. 5 Individual cells. Supporting cells should be used within 5 days from the day after inoculation.

[0143] (Introducing mutations into chicken epiSC)

[0144] Germinal disc cells isolated from one embryo were cultured in a single culture dish inoculated with supporting cells. The composition of the culture medium used for culturing germinal disc cells is shown in Table 1. In addition, the medium was based on KnockOut-DMEM in Table 1, with recombinant chicken leukemia inhibitory factor (recombinant chicken LIF) added to the heated minimum required amount of the medium just before use.

[0145] The preparation method of recombinant chicken LIF is described here. Chicken embryo cell line CHCC-OU2 was cultured in 5% CO2 at 37°C using low-glucose DMEM containing 10% FBS (Hyclone; Thermo Fisher Scientific), 100 μg / ml penicillin, and 70 μg / ml streptomycin. The coding region of LIF was amplified by PCR using the forward primer shown in sequence number 32 and the reverse primer shown in sequence number 33. The PCR product was treated with Nhe I and Sal I and subcloned into the histidine-tagged pSecTag2A plasmid (Invitrogen).

[0146] Next, the myc-determinant site was removed from the pSecTag2A plasmid using restriction enzymes. The recombinant plasmid was introduced into CHCC-OU2 cells using Polyfect transfection reagent (Qiagen), and cells were screened using medium containing 0.25 μg / ml Zeocin (Invitrogen). Stable cell lines secreting LIF with biological activity were screened, and recombinant chicken LIF was purified from the culture supernatant using ProBond resin (Invitrogen).

[0147] [Table 1]

[0148] Reagent Name (Manufacturer, Catalog Number #) Final concentration KnockOut serum alternative (Invitrogen #10828-028) 20% Chicken serum (Invitrogen #16110-082) 2% Sodium pyruvate (Gibco #11360-070) 1% MEM NEAA (Gibco #11140-050) 1% GlutaMax (Gibco #35050-061) 1% 100X Nucleoside (Millipore #ES-008D) 1% A stock solution containing a mixture of antibacterial and antifungal agents (nacalai tesque #09366-44) 1% β-Mercaptoethanol (Sigma #M7522) 0.1mM KnockOut-DMEM (Invitrogen #10829-018) Recombinant chicken LIF 20ng / mL

[0149] Using FuGENE HD (manufactured by Promega), 6.5 μg of the G-TALEN expression vector was introduced into epiSCs obtained by culturing germinal disc cells on Supporting cells for 2–3 days. 24 hours after G-TALEN expression vector introduction, puromycin was added to the culture medium at a concentration of 2 μg / mL, and the cells were cultured for 48 hours. After 48 hours, the culture medium was exchanged to remove puromycin, and the cells were cultured for approximately 10 days until the epiSCs proliferated stably.

[0150] Genomic DNA was recovered from stably proliferating epiSCs using the DNeasy Blood & Tissue Kit (QIAGEN). Genomic PCR was performed under the following conditions: 35 cycles of 94°C for 2 minutes, followed by 98°C for 10 seconds, 68°C for 30 seconds, and 72°C for 2 minutes. The nucleotide sequences of the forward and reverse primers used in the genomic PCR are shown in SEQ ID NO. 34 and SEQ ID NO. 35, respectively.

[0151] (Cel-I determination)

[0152] Next, the PCR products were rehybridized and treated with Surveyor nuclease for Cel-I assay at the heteroduplex cleavage site. The Cel-I assay used a SURVEYOR mutation detection kit (manufactured by Transgenomic). The PCR products were purified using a Wizard SV Gel and a PCR Clean-up system (manufactured by Promega). DNA was eluted using 15 μl of the solution, and the DNA concentration was quantified after elution.

[0153] Next, prepare the DNA solution for Cel-I assay according to the following composition.

[0154] PCR product 400ng

[0155] 10× Hybridization Buffer (100mM Tris-HCl (pH 8.5), 750mM KCl, 15mM MgCl2) 0.8μl

[0156] Prepare 8 μl using sterile distilled water.

[0157] The DNA solution was kept at 95°C for 5 minutes and then cooled to 25°C over 60–90 minutes.

[0158] Add 0.4 μl of enhancer S and 0.4 μl of nuclease S to the DNA solution, transfer thoroughly, and incubate at 42 °C for 30 minutes. Immediately after the reaction, perform electrophoresis on agarose gel or polyacrylamide gel.

[0159] (result)

[0160] In Cel-I assays, no clear bands indicating mutation introduction were obtained. On the other hand, when analyzing the nucleotide sequences of the PCR products, such as... Figure 4 As shown, two mutations were identified in the target region: the addition of one base and the substitution of one base (see underlined). However, none of these mutations produced a stop codon.

[0161] Example 3: Preparation and evaluation of cleavage activity of platinum-based TALEN expression vector

[0162] A highly active TALEN targeting the nucleotide sequence of exon 1, namely platinum-gate TALEN (hereinafter referred to as "P-TALEN"), was constructed. A module capable of binding to the effector sequence in exon 1 was constructed using the 6-module assembly method, and P-TALEN expression vectors (left and right) were fabricated relative to exon 1. The P-TALEN expression vectors were prepared using the platinum-gate TALEN kit and the Yamamoto Lab TALEN Accessory Pack (both available from Addgene) according to the instructions provided with the kit. The cleavage activity of the P-TALEN expression vectors was evaluated by SSA assay in the same manner as described above.

[0163] (result)

[0164] Figure 5 The cleavage activity of the P-TALEN expression vector obtained by SSA assay is shown. The cleavage activity of the P-TALEN expression vector exceeding that of the G-TALEN expression vector targeting exon 3 prepared in Example 1 was confirmed.

[0165] Example 4: Single vectorization of P-TALEN and introduction of mutations into chicken epiSC

[0166] To improve the delivery efficiency of the P-TALEN expression vector, the two vectors, P-TALEN expression vector (left) and P-TALEN expression vector (right), were converted into a single vector. At the same time, the expression cassette of the puromycin resistance gene was introduced into this single vector to instantly concentrate the cells containing the vector. Figure 6 The composition of the constructed single vector is shown. The cleavage activity of the constructed single vector was evaluated by SSA assay in the same manner as described above. Furthermore, chicken embryo fibroblasts (CEFs) were used in the SSA assay to evaluate the cleavage activity within chicken cells.

[0167] 6.5 μg of a single vector was introduced into cultured epiSCs using FuGENE HD (manufactured by Promega). In addition, to improve mutation introduction efficiency, the single vector and chicken exonuclease I expression vector (EXO I) were simultaneously introduced into epiSCs, independent of the single vector introduction alone. Twenty-four hours after single vector introduction, puromycin was added to the culture medium at a concentration of 2 μg / mL, and the cells were cultured for 48 hours. After 48 hours, the culture medium was exchanged to remove puromycin, and the cells were cultured for approximately 10 days until the epiSCs stably proliferated.

[0168] Genomic DNA was recovered from epiSC in the same manner as described above for genomic PCR. The nucleotide sequences of the forward and reverse primers used in the genomic PCR are shown in SEQ ID NO. 36 and SEQ ID NO. 37, respectively. The PCR products were then used for Cel-I assay.

[0169] (result)

[0170] Figure 7 This displays the cleavage activity of the single vector obtained by SSA assay. Relative activity refers to the value measured when the single vector is introduced into a CEF containing no target sequence, with 1 being the relative value. For example... Figure 7 As shown, the single vector exhibits sufficient cleavage activity. Furthermore, "CMV-ptTALEN L+R" represents a vector where the expression of left and right TALENs is controlled by the CMV promoter, and "CAG-ptTALEN L+R" represents a vector where the expression of left and right TALENs is controlled by the CAG promoter. In genomic PCR, as... Figure 8 As shown in (A), a shift band was observed, which indicates a heteroduplex produced by mutation in the drug screening system. In Cel-I assays, as... Figure 8 As shown in (B), banding of the digested fragments was observed, indicating the introduction of mutations from the epiSC genome, which underwent drug screening. The effect of chicken EXO I introduction was not confirmed.

[0171] Example 5: Importing knockout mutations and clones into epiSC

[0172] The mutation-introducing region of the epiSC genomic DNA was amplified by PCR, cloned into a vector, and its nucleotide sequence was analyzed. Forty-three clones were analyzed, with the following results: 10 clones with deletions, 1 clone with insertions, and 2 clones with substitutions. The mutation introduction efficiency was as high as 30%. In the analysis of knockout mutations, knockout mutations resulting from the insertion of a stop codon were detected in 3 clones (2 clones with deletions and 1 clone with insertions). Figure 9 In the example, #3 (A) and #36 (C) with deletions and #4 and #18 (B) with insertions are shown as representative nucleotide sequences with introduced mutations. Figure 9 In the diagram, double underlines for wild-type (D) indicate the signal sequence, while underlines for #3, #18, and #36 indicate the mutated amino acid sequences resulting from the introduction of the mutation. The knockout mutation efficiency was 7%.

[0173] After introducing a single vector into epiSC, 3300 cells were seeded in 96-well plates for cloning. EpiSC colonies multiplied from 49 wells across 9 plates, ultimately successfully multiplying in 27 wells. While these 27 wells were cryopreserved, a subset of cells was used to extract the genome, which was then analyzed using CE1 sequencing in the same manner as described above, followed by nucleotide sequence analysis.

[0174] (result)

[0175] Of the 27 wells, 4 wells confirmed a positive result for the Cel-I assay. Figure 10 After stabilizing the cell proliferation as illustrated in clone #5, the nucleotide sequence was analyzed. Figure 11 As shown, in the nucleotide sequence of the mutant introduction region of the cloned ovomucoid knockout epiSC strain #4, a "T" is inserted at the position following the 35th base starting from the 5' end. Converting this nucleotide sequence to an amino acid sequence reveals that stop codons are inserted at the positions corresponding to the 26th and 31st residues starting from the N-terminus.

[0176] On the other hand, in the nucleotide sequence of the mutant introduction region of the ovomucoid knockout epiSC strain #5, in relation to... Figure 9 The nucleotide sequence shown in (C) has 5 bases missing at the same positions. When this nucleotide sequence is converted into an amino acid sequence, it can be seen that stop codons are inserted at the positions corresponding to the 24th and 29th residues starting from the N-terminus.

[0177] When the amino acid sequence encoded by the nucleotide sequence of epiSC strain #4 is compared with the amino acid sequence of wild-type ovomucoid, it can be seen that: Figure 12 As shown in (A), an amino acid mutation was performed by frameshifting, starting from the 13th residue from the N-terminus of the signal peptide, and the amino acid sequence was translated up to the 25th residue. Since the signal peptide was cleaved in the endoplasmic reticulum and not secreted, this mutation was confirmed to be a knockout mutation of ovomucoid.

[0178] When the amino acid sequence encoded by the nucleotide sequence of epiSC strain #5 is compared with the amino acid sequence of wild-type ovomucoid, it can be seen that: Figure 12 As shown in (B), a frameshift mutation was performed starting from the 11th residue from the N-terminus of the signal peptide, and translation was completed up to the 23rd residue. EpiSC strain #5 was also confirmed to be a knockout mutation of ovomucoid. Furthermore, analysis of the nucleotide sequence of the ovomucoid knockout epiSC strain #5 confirmed complete cloning as no other nucleotide sequences were identified. As a precaution, cloning was performed again, and ovomucoid knockout epiSC strain #5-3 was also prepared.

[0179] Example 6: Production of chimeric chickens

[0180] Egg mucin knockout epiSC strains #4, #5, and #5-3 were transplanted into the blastodiscs of newly laid fertilized egg embryos irradiated with 5 Gy of gamma rays, and the germ cell chimeric chickens (G0) were hatched.

[0181] (result)

[0182] Eighteen chimeric chickens were produced. Since the oocyte mucin knockout epiSC strain is a striped Plymouth Rock breed (chicks with black feathers) and the recipient embryo used for transplantation is a white Leyhorn breed (chicks with white feathers), when it is a chimera, the oocyte mucin knockout epiSC differentiates into the epidermis, and black feathers can be identified. Figure 13 (A) and (B) show the appearance of chimeric chickens from epiSC strain #5 and #4, respectively. Black feathers were confirmed in these chimeric chickens. Details of the chimeric chickens are as follows, as shown in Table 2: 6 males, 7 females, and 5 unidentified individuals. Eleven out of the 18 chimeric chickens were black-feathered. The "Feathers" section of Table 2 shows the proportion of black feathers.

[0183] [Table 2]

[0184] Serial Number Individual ID hatch day epiSC male and female feather 1 1 20140930 #5 ♂ Black (15%) 2 2 20141017 #5 ♂ Black (5%) 3 7289 20141111 #5 ♂ - 4 7292 20141230 #5-3 ♀ Black (5%) 5 7293 20150106 #5 ♀ Black (1%) 6 7294 20150407 #5 ♂ Black (30%) 7 7295 20150407 #5 ♀ - 8 7296 20150407 #5 ♂ - 9 7606 20150414 #5 ♀ Black (10%) 10 7607 20150414 #5 ♂ - 11 7608 20150414 #5-3 ♀ black(%) 12 7609 20150414 #5-3 ♀ - 13 7610 20150414 #5-3 ♀ - 14 3 20150519 #5 Undecided - 15 4 20150519 #5-3 Undecided Black (5%) 16 5 20150609 #5 Undecided Black (15%) 17 6 20150609 #5-3 Undecided Black (50%) 18 7 20150616 #4 Undecided Black (>90%)

[0185] By mating the resulting chimeric chickens with males and females, it is possible to produce a homozygous chicken (G1) with the ovomucin gene knocked out. Because the ovomucin gene is knocked out in this chicken, the eggs it lays do not contain ovomucin.

[0186] Example 7: Construction of CRISPR / Cas9 vectors

[0187] To construct the CRISPR / Cas9 vector, as shown below, similar to the TALEN case, a puromycin resistance gene was inserted into the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (manufactured by Addgene). (See reference...) Figure 14 ).

[0188] First, target sequences capable of inducing the knockout of the ovomucoid gene were detected using CRISPR Direct (http: / / crispr.dbcls.jp / ). The search results identified target sequences at four sites in exon 1 and two sites in exon 2 (sequence numbers 8–13). Based on these target sequences, [the following is a list of synthesized products / technologies]. Figure 15 The oligoDNA with the shown nucleotide sequence. In Figure 15In this study, "justice" targets the positive strand of the ovomucoid gene, while "antisense" targets the negative strand of the ovomucoid gene. Figure 15 The underlined nucleotide sequences show the added sequences used for insertion into the vector.

[0189] The synthesized oligoDNA was introduced into vectors to create six CRISPR / Cas9 vectors (CRISPR / Cas9-Pur) for ovomucoid knockout. Additionally, reporter vectors for SSA assays were created in the same manner as described above, based on the target sequences, and the cleavage activity of the target sequences was evaluated. Furthermore, regarding exon 1, the nucleotide sequences of the sense and antisense oligoDNA contained in the synthetic oligoDNA inserted into the reporter vector for SSA assays are shown in SEQ ID NO. 38 and SEQ ID NO. 39, respectively. Furthermore, regarding exon 3, the nucleotide sequences of the sense and antisense oligoDNA contained in the aforementioned synthetic oligoDNA inserted are shown in SEQ ID NO. 40 and SEQ ID NO. 41, respectively.

[0190] (Study on the cleavage activity of CRISPR / Cas9)

[0191] To determine the cleavage activity of the target sequence of the CRISPR / Cas9 vector used for ovomucoid knockout, the CRISPR / Cas9 vector was introduced into HEK293 cells, followed by SSA assays. Additionally, to test the cleavage activity in chicken cells, the same SSA assay was performed using epiSC.

[0192] (result)

[0193] It can be seen that: Figure 16 As shown, the two vectors (exon 1 #1 and exon 1 #2) prepared using exon 1 as a target exhibit approximately twice the cleavage activity of the CMV-ptTALEN L+R vector, which showed high activity in SSA assays. This indicates that... Figure 17 As shown, the #1 vector exhibits the same cleavage activity as TALEN even in epiSC.

[0194] This embodiment demonstrates that even using CRISPR / Cas9, the ovomucoid locus in chicken epiSC can be modified. Furthermore, using CRISPR / Cas9, chickens with the ovomucoid gene knocked out can be produced.

[0195] The above embodiments are for illustrative purposes only and do not limit the scope of the invention. That is, the scope of the invention is shown not by the embodiments, but by the scope of the claims. Furthermore, various modifications made within the scope of the claims and their equivalent inventive meaning are also considered to be within the scope of the invention.

[0196] This application is based on Japanese Patent Application No. 2015-168372, filed on August 27, 2015. The description, claims, and drawings of Japanese Patent Application No. 2015-168372 are incorporated herein by reference in their entirety.

[0197] Industrial practicality

[0198] This invention is suitable for the manufacture of bird eggs, especially chicken eggs.

Claims

1. A method for producing birds, comprising the following steps: The modification step involves cleaving and modifying the oval mucin locus of avian cells with pluripotency and germ cell differentiation capacity using a transcription activator-like effector nuclease; and The transplantation step involves transplanting the cells modified with the oval mucin gene locus into the embryo of a bird. The transcription activator-like effector nuclease comprises a first nuclease and a second nuclease, wherein the first nuclease contains the amino acid sequence shown in sequence number 4, and the second nuclease contains the amino acid sequence shown in sequence number 5. In the modification step, A single vector, obtained by combining the vector expressing the first nuclease and the vector expressing the second nuclease into a single vector, is introduced into the cell.

2. A method for producing birds, comprising the following steps: The modification step involves cleaving and modifying the oval mucin locus of avian cells with pluripotency and germ cell differentiation capacity using a transcription activator-like effector nuclease; and The transplantation step involves transplanting the cells modified with the oval mucin gene locus into the embryo of a bird. The transcription activator effector nucleases are a first nuclease and a second nuclease, wherein the first nuclease contains the amino acid sequence shown in sequence number 4, and the second nuclease contains the amino acid sequence shown in sequence number 5.

3. The method for producing birds according to claim 1 or 2, wherein, In the transplantation step, The cells with mutations in the signal sequence of the oocyte mucin gene locus are transplanted into the embryo.

4. The method for producing birds according to claim 1 or 2, wherein, In the transplantation step, The cell containing the stop codon in the third exon, counting from the 5' end of the oocyte mucin gene locus, is transplanted into the embryo.

5. The method for producing birds according to claim 1 or 2, wherein, It also includes the following steps: The incubation step involves incubating an egg containing an embryo from which the cell was transplanted in the transplantation step, thereby producing a chimeric individual. The production step involves mating the chimeric individuals to produce birds with genomes containing the modified ovomucin locus through homologous conjugation.

6. A method for manufacturing an egg, wherein, Including the bird production method as described in claim 1 or 2, This includes obtaining eggs laid by birds produced using the bird production method described above.

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