Methods for producing birds and methods for producing eggs

Abstract

To provide a method for making birds which can sufficiently reduce allergenicity of eggs, and a method for producing eggs with sufficiently reduced allergenicity.SOLUTION: A method for making birds includes: a modification step of cleaving an ovomucoid gene locus of a bird cell having pluripotency or germinal differentiation capacity with a transcription activator-like effector nuclease, thereby modifying the ovomucoid gene locus; and a transplantation step of transplanting the cell with the modified ovomucoid gene locus into a bird's embryo. The transcription activator-like effector nuclease comprises a first nuclease and a second nuclease. In the modification step, a vector expressing the first nuclease and a vector expressing the second nuclease are integrated into a single vector, referred to as a 'one-vector', which is incorporated into the cell.SELECTED DRAWING: Figure 13

Description

Technical Field

[0001] The present invention relates to a method for producing birds and a method for producing eggs.

Background Art

[0002] Chicken eggs are the number one cause of food allergies among Japanese people. Some proteins in chicken eggs become allergens that cause food allergies. Examples of allergens contained in chicken eggs include ovomucoid, ovalbumin, lysozyme, and ovotransferrin.

[0003] In order not to cause food allergies caused by chicken eggs, attempts have been made to remove the above-mentioned allergens from chicken eggs. To remove allergens from chicken eggs, it is sufficient to produce chickens in which the genes encoding the allergens are disrupted.

[0004] Non-Patent Document 1 discloses a genetically modified chicken in which the ovalbumin gene has been knocked out with transcription activator-like effector nuclease (TALEN). According to the genetically modified chicken, it is considered that chicken eggs containing no ovalbumin can be obtained.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] Even in chicken eggs that do not contain ovalbumin, the egg white contains about 10% ovomucoid. Ovomucoid is the most allergenic protein in chicken eggs. Because ovomucoid has high physicochemical stability, its allergenicity is maintained even when heated. Therefore, even if chicken eggs are made free of ovalbumin or ovalbumin is inactivated by heating using the method disclosed in Non-Patent Document 1, it is difficult to say that the allergenicity of the chicken eggs has been sufficiently reduced.

[0007] Furthermore, Non-Patent Document 1 mentioned above uses primordial germ cells. Since primordial germ cells are present in only a small number during development, techniques for culturing them are necessary. Several methods for culturing primordial germ cells have been reported to date, but these reports are limited to a few research institutions, and even when culturing according to the reported methods, it is not always possible to successfully culture them, resulting in a lack of reliability.

[0008] This invention has been made in view of the above circumstances, and aims to provide a method for producing birds that can sufficiently reduce the allergenicity of eggs, and a method for producing eggs with sufficiently reduced allergenicity. [Means for solving the problem]

[0009] The inventors knocked out the ovomucoid gene in chickens using a conventional homologous recombination method. However, conventional homologous recombination methods require long-term cell culture, resulting in cell damage and abnormal karyotypes. Furthermore, even when the chimeric chickens obtained by homologous recombination were crossbred, homozygous chickens with knocked-out ovomucoid genes were not obtained. The eggs laid by these chimeric chickens contained ovomucoid derived from the recipient's genome, and the allergenicity of the eggs was not sufficiently reduced.

[0010] Therefore, the inventors diligently conducted research and completed the present invention by applying genome editing technology. That is, A method for producing birds according to the first aspect of the present invention is: A modification step involves cleaving and modifying the ovomucoid gene locus of avian cells possessing pluripotency and germ cell differentiation ability using a transcription activator-like effector nuclease, A transplantation step involves transplanting the cells, which have been modified at the ovomucoid gene locus, into a bird embryo. Includes, The aforementioned transcription activator-like effector nuclease is The amino acid sequence shown in Sequence ID No. 4 The first nuclease and The amino acid sequence shown in Sequence ID No. 5 It is a second nuclease, In the aforementioned modification step, A one-vector, which combines a vector expressing the first nuclease and a vector expressing the second nuclease into a single vector, is introduced into the cells.

[0011] A method for producing birds according to a second aspect of the present invention is: The ovomucoid gene locus in avian cells possessing pluripotency and germ cell differentiation ability is cleaved and modified using a transcription activator-like effector nuclease. Modification Steps and , Modified the ovomucoid gene locus. The aforementioned cells birds of Embryo transfer do Transplantation steps and , Includes, The aforementioned transcription activator-like effector nuclease is The first nuclease consists of the amino acid sequence shown in SEQ ID NO: 4, and the second nuclease consists of the amino acid sequence shown in SEQ ID NO: 5. .

[0012] Furthermore, in the transplantation step, The cells containing a mutation in the signal sequence of the ovomucoid gene locus are transplanted into the embryo. It would be acceptable to do so.

[0013] Furthermore, in the transplantation step, The cells containing a stop codon in the third exon from the 5' end of the ovomucoid locus are transplanted into the embryo. It would be acceptable to do so.

[0015] Also, an incubation step of incubating an egg containing an embryo transplanted with the cells in the transplantation step to produce a chimeric individual, A production step of producing a bird having a genome homozygous for the modified ovomucoid locus by mating the chimeric individuals, Further comprising It may be.

[0016] The method for producing an egg according to the third aspect of the present invention is The method for producing a bird according to the first or second aspect of the present invention described above Includes, Method for producing the aforementioned birds Including obtaining an egg laid by a bird produced by

Effects of the Invention

[0017] According to the present invention, the allergenicity of eggs can be sufficiently reduced. Further, according to the present invention, eggs with sufficiently reduced allergenicity can be obtained.

Brief Description of Drawings

[0018] [Figure 1] It is a diagram showing the nucleotide sequences of the first exon (exon 1), the second exon (exon 2), and the third exon (exon 3) counted from the 5'-end at the ovomucoid locus of the chicken. [Figure 2] It is a diagram showing the TALEN recognition regions identified in exon 1 and exon 3. [Figure 3] It is a diagram showing the relative cleavage activities of TALEN targeting exon 1 and exon 3. [Figure 4] It is a diagram showing the nucleotide sequence of the target region in the genome of pluripotent stem cells derived from the upper layer of the blastoderm into which the TALEN expression vector targeting exon 3 was introduced. [Figure 5] It is a diagram showing the relative cleavage activities of TALEN targeting exon 3 and highly active TALEN targeting exon 1. [Figure 6]This diagram shows the structure of a TALEN expression vector. [Figure 7] This figure shows the relative cleavage activity of TALENs within chicken cells. [Figure 8] This figure shows the introduction of mutations into pluripotent stem cells derived from the upper layer of the blastocyst using a TALEN expression vector. (A) shows the results of genomic PCR (polymerase chain reaction). (B) shows the results of the Cel-I assay. [Figure 9] This figure shows a portion of the ovomucoid locus nucleotide sequence of the clone and the amino acid sequence encoded by that nucleotide sequence. (A), (B), and (C) show clones in which the knockout mutation was obtained. (D) shows the wild type. [Figure 10] This figure shows colonies of pluripotent stem cell lines derived from the upper blast layer containing cloned knockout mutations. [Figure 11] This figure shows a portion of the ovomucoid locus nucleotide sequence in a pluripotent stem cell line derived from the upper blast layer containing a cloned knockout mutation, as well as in the wild type. [Figure 12] This figure shows a portion of the nucleotide sequence of the ovomucoid locus of a pluripotent stem cell line derived from the epiblast layer containing a cloned knockout mutation, as well as 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 sequence and amino acid sequence of pluripotent stem cell line #4 derived from the epiblast layer. (B) shows the nucleotide sequence and amino acid sequence of pluripotent stem cell lines #5 and #5-3 derived from the epiblast layer. [Figure 13] This figure shows photographs of knockout chimeric chickens produced from pluripotent stem cell lines derived from the blastodisc epilayer containing the knockout mutation. (A) shows a chimeric chicken derived from pluripotent stem cell line #5 derived from the blastodisc epilayer. (B) shows a chimeric chicken derived from pluripotent stem cell line #4 derived from the blastodisc epilayer. [Figure 14] This figure shows the configuration of a CRISPR / Cas9 vector for ovomucoid knockout. [Figure 15]This figure shows the base sequences of each oligoDNA incorporated into the ovomucoid knockout CRISPR / Cas9 vector. [Figure 16] This figure shows the relative cleavage activity of the ovomucoid knockout CRISPR / Cas9 vector in HEK293 cells. [Figure 17] This figure shows the relative cleavage activity of the ovomucoid knockout CRISPR / Cas9 vector in pluripotent stem cells derived from the upper layer of the blast. [Modes for carrying out the invention]

[0019] Embodiments of the present invention will now be described. However, the present invention is not limited to the embodiments and drawings described below.

[0020] (Embodiment 1) First, Embodiment 1 will be described. The bird according to Embodiment 1 does not contain artificially introduced foreign genes in its genome and lays eggs in which the ovomucoid content is reduced compared to that of the wild type.

[0021] The birds are not particularly limited and include, for example, chickens, ducks, turkeys, waterfowl, geese, quail, pheasants, parrots, finches, hawks, ostriches, emus, and cassowaries. Preferably, the birds are chickens. The breeds of chickens are not particularly limited and include, for example, White Leghorn, Brown Leghorn, Barred Rock, Sussex, New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray, Italian Partidge colored, and Korean Oge.

[0022] The avian genome according to this embodiment does not contain any artificially introduced foreign genes. Here, "artificially introduced foreign genes" refers to mutated genes and genes not naturally present in the avian genome, which are artificially introduced using techniques such as genetic engineering. Methods for artificially introducing foreign genes into the genome include homologous recombination, retroviral vectors, lentiviral vectors, and artificial viral vectors. The avian genome described above does not contain any foreign genes introduced by these methods.

[0023] Ovomucoid is a heat-stable glycoprotein with a molecular weight of approximately 28,000. It is produced by secretory cells in the oviduct. Ovomucoid is usually found mainly in the egg white of bird eggs. For example, ovomucoid accounts for about 11% by weight of the protein in the egg white of a chicken egg. The ovomucid content in eggs laid by the birds according to this embodiment is reduced compared to the eggs of wild-type birds of the same species. Here, "wild-type" refers to the same species of bird as described above, but without artificial genetic modification.

[0024] The ovomucoid content in eggs can be quantified using known methods for detecting target proteins. For example, samples prepared from egg whites collected from wild-type eggs and eggs laid by the birds according to this embodiment can be immunostained using an antibody that binds to ovomucoid in a Western blot, and the ovomucoid content can be compared based on the intensity of the bands.

[0025] To ensure quantitative accuracy, the ovomucoid content is preferably measured using sandwich ELISA (Enzyme-Linked ImmunoSorbent Assay). The capture antibody and detection antibody used in sandwich ELISA may be monoclonal or polyclonal antibodies. For example, rabbit anti-ovomucoid antibody and mouse anti-ovomucoid antibody can be used as the capture antibody and detection antibody, respectively.

[0026] For rabbit anti-ovomucoid antibodies, polyclonal antibodies can be used, which are obtained by recovering antiserum from rabbits immunized with ovomucoid and purifying them by affinity chromatography using ovomucoid. On the other hand, for mouse anti-ovomucoid antibodies, monoclonal antibody-producing hybridomas can be established using a cell fusion method with mouse spleen cells, and mouse anti-ovomucoid antibodies can be used, which are purified from ascites fluid antibodies. The detection antibody is not particularly limited, but it should be labeled with peroxidase. When labeled with peroxidase, ovomucoid can be quantified by the color development of TMB (3,3',5,5'-tetramethylbenzidine). The ovomucoid content may also be evaluated by its concentration in the sample. By constructing an appropriate sandwich ELISA, ovomucoid can be quantified with a detection limit of about 50 pg / ml.

[0027] As described above, by quantifying the ovomucoid content, it can be confirmed that the ovomucoid content in the eggs laid by the birds according to this embodiment is reduced compared to that of the wild type of the same bird species. For example, the ovomucoid content in the eggs laid by the birds according to 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 ovomucoid content in the eggs of the wild type of the same bird species. In addition, when ovomucoid is quantified in the eggs laid by the birds according to this embodiment, the ovomucoid concentration may be below the detection limit. In particular, if the bird according to this embodiment is a chicken, the ovomucoid content in the egg white protein of the chicken eggs laid by the chicken may be 0-8% by weight, 0-4% by weight, 0-3% by weight, 0-2% by weight, or 0-1% by weight. The ovomucoid content may also be evaluated by the weight of ovomucoid per unit volume of egg white.

[0028] Particularly preferably, the eggs laid by the birds according to this embodiment do not contain ovomucoid. Note that "ovomucoid-free" means that the eggs do not contain ovomucoid, which is present in the egg white of wild-type eggs. Therefore, eggs that do not contain ovomucoid also include eggs that contain fragments of ovomucoid that are not the full length.

[0029] In the avian genome according to this embodiment, the ovomucoid locus contains a knockout mutation that prevents ovomucoid expression, or a mutation that prevents the expression of the full-length ovomucoid, resulting in abnormal ovomucoid expression. While any mutation is desirable as long as ovomucoid is not expressed normally, specifically, the insertion of a stop codon into an exon within the ovomucoid locus is preferred.

[0030] The ovomucoid locus contains five exons. If we number the exons 1-5 sequentially from the 5' end, the stop codon can be inserted at any position within exons 1-5. More preferably, the birds described above contain a stop codon in at least one exon between exons 1-3 of the ovomucoid locus. If a stop codon is inserted in exons 1-5 within the ovomucoid locus, the ovomucoid is not fully synthesized, and full-length ovomucoid is not expressed. Depending on the position of the stop codon insertion, fragments of the ovomucoid may be expressed. However, if the antigenic epitopes of these ovomucoid fragments are reduced compared to full-length ovomucoid, allergenicity can be decreased.

[0031] Furthermore, the mutation may also be a mutation in the start codon within the ovomucoid locus. If there is a mutation in the start codon within the ovomucoid locus, mRNA-based ovomucoid synthesis will not occur. For example, the birds mentioned above do not contain a start codon in exon 1 of the ovomucoid locus.

[0032] Preferably, the above-mentioned birds have a modified signal sequence at the ovomucoid locus. The signal sequence at the ovomucoid locus includes 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 shows the nucleotide sequences of exon 1, exon 2, and exon 3. In Figure 1, the nucleotides of exons are shown in uppercase, and the nucleotides of introns are shown in lowercase. The underlined nucleotide sequence shown in Figure 1 is the signal sequence. In order to prevent the expression of not only the full-length ovomucoid but also fragments of the ovomucoid, the modification of the signal sequence may involve introducing a mutation in the start codon ATG, or introducing a mutation that produces a stop codon in the signal sequence. Preferably, the stop codon is contained in exon 1 of the ovomucoid locus. The nucleotide sequences of exon 1, exon 2, and exon 3 are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0033] As mentioned above, the birds described above do not contain any artificially introduced foreign genes in their genomes. Therefore, in creating the birds described above with a modified ovomucoid locus, it is preferable to use genome editing technology, which will be detailed below, rather than homologous recombination, which replaces a specific gene on the genome with a foreign gene.

[0034] As explained in detail above, the birds according to this embodiment do not express ovomucoid normally, and therefore lay eggs with a lower ovomucoid content than wild-type eggs. Ovomucoid is so allergenic that in egg allergy tests, it is tested independently in addition to egg yolk and egg white. Therefore, eggs with a lower ovomucoid content than wild-type eggs can sufficiently reduce the allergenicity of the eggs.

[0035] Furthermore, the aforementioned birds do not contain artificially introduced foreign genes in their genomes. The absence of foreign genes prevents the emergence of unexpected phenotypes and toxicity. Additionally, the absence of foreign genes minimizes any potential compromise to the reliability of reproductive genes in these birds.

[0036] Furthermore, because ovomucoid has high physicochemical stability, its allergenicity is maintained even in processed foods or vaccines containing heat-treated bird egg whites. Therefore, eggs with a reduced ovomucoid content compared to wild-type eggs according to this embodiment are useful as raw materials for various products such as processed foods and vaccines.

[0037] Furthermore, the birds described above may contain a stop codon in at least one exon of exons 1-3 of the ovomucoid locus. If a stop codon is contained in exon 3, the antigenic epitopes of the secreted ovomucoid fragments will be less than those of full-length ovomucoid, thus reducing allergenicity. In particular, by containing a stop codon in exon 1 of the ovomucoid locus, the birds according to this embodiment can lay eggs that do not contain any ovomucoid fragments, let alone full-length ovomucoid. Also, if a start codon is not contained in exon 1 of the ovomucoid locus, mRNA-based ovomucoid synthesis does not occur, so the birds can lay eggs that do not contain ovomucoid.

[0038] Furthermore, the above-mentioned birds may have modified signal sequences at the ovomucoid locus. Since the signal peptide corresponding to the signal sequence is cleaved within the endoplasmic reticulum of the cell and not secreted, if the signal sequence contains a stop codon, the secretion of peptides originating from the ovomucoid locus into egg white can be prevented. As a result, the allergenicity caused by ovomucoid can be further reduced.

[0039] In this embodiment, the birds are preferably chickens. Chickens lay eggs, which are in high demand, and can efficiently supply eggs with sufficiently reduced allergenicity. In terms of supplying eggs for consumption, quail and other birds are also preferable to chickens.

[0040] Furthermore, mutations in the ovomucoid locus may include mutations that cause frameshifts to exons within the ovomucoid locus, among others.

[0041] In another embodiment, bird eggs are provided that do not contain artificially introduced foreign genes in their genome and have a reduced ovomucoid content compared to wild-type eggs. Chicken eggs, in particular, are widely used as raw materials for confectionery, beverages, processed foods, etc., or for the manufacture of pharmaceuticals such as vaccines. By using chicken eggs according to this embodiment, even if ovomucoid is mixed into various products, the allergenicity of ovomucoid can be reduced. Furthermore, by using ovomucoid-free bird eggs, the risk of the most allergenic ovomucoid being mixed into various products can be minimized.

[0042] (Embodiment 2) Next, Embodiment 2 will be described. Embodiment 2 describes a method for producing birds suitable for the birds described in Embodiment 1.

[0043] To create birds that lay eggs with a reduced ovomucoid content compared to wild-type birds, it is necessary to create birds with a modified ovomucoid gene locus. To create genetically modified animals, the genome of a one-cell stage fertilized egg must be modified, and individuals with the desired mutation must be selected from the resulting individuals. Therefore, in creating genetically modified animals, it is necessary to manipulate fertilized eggs in vitro and develop individuals from them. For example, in mice and rats, it is possible to obtain multiple unfertilized eggs from a single female. Thus, after in vitro fertilization, it is possible to develop the eggs to the one-cell stage, modify their genomes, and then return the fertilized eggs to the female's ovaries to create multiple individuals.

[0044] On the other hand, in vitro fertilization techniques have not been established in birds. Furthermore, only one single-cell stage fertilized egg can be obtained from each egg-laying bird. In addition, it is difficult to identify single-cell stage fertilized eggs present in the oviduct. For these reasons, applying genetic modification technology to avian fertilized eggs is difficult. Therefore, to create genetically modified birds, pluripotent stem cells with pluripotency or germ cell differentiation ability are used instead of fertilized eggs.

[0045] Therefore, the method for producing birds according to this embodiment includes a modification step of cutting and modifying the ovomucoid gene locus of avian pluripotent stem cells with a programmable endonuclease, and a transplantation step of transplanting the pluripotent stem cells with the modified ovomucoid gene locus into an avian embryo.

[0046] First, the above modification steps will be explained in detail. Examples of pluripotent stem cells in birds include embryonic stem cells (ES cells) that possess pluripotency and germ cell differentiation ability. Bird ES cells are, for example, epiblast-derived stem cells (hereinafter simply referred to as "epiSC") that can be established from blast cells isolated from the epiblast of a fertilized egg. In the case of chickens, the blast of stage X of the Eyal-Giladi and Kochav developmental stages (I-XIV) consists of the epiblast. Chicken epiSCs are obtained by culturing blast cells isolated from the epiblast using known methods on supporting cells whose cell proliferation has been stopped, for example, by irradiation or mitomycin C treatment. Examples of supporting cells include chicken embryonic fibroblasts, mouse embryonic fibroblasts, and mouse embryonic fibroblast-derived cell lines.

[0047] Furthermore, primordial germ cells may be used because they possess pluripotency and germ cell differentiation ability. Primordial germ cells can be isolated, for example, from the fetal gonads of birds using known methods. Below, we will describe the case in which epiSCs are used in the modification step.

[0048] In ovomucoid locus modification, a programmable endonuclease is used to cleave the ovomucoid locus on the genome, for example, in epiSC, at a specific site. Programmable endonucleases are used in so-called genome editing technologies that can specifically modify (delete, replace, or insert) target sites in the genome. Programmable endonucleases are designed according to the base sequence of the target DNA and can cleave DNA at any base sequence. Programmable endonucleases are not limited to, but include, for example, TALENs, 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").

[0049] TALENs and ZFNs are polypeptides consisting of a DNA-binding domain and a DNA-cleaving domain. TALENs and ZFNs cleave double-stranded DNA by forming a dimer at the binding site of the DNA-binding domain, where a pair of DNA-cleaving domains meet in close proximity. The DNA-binding domain contains multiple repeating DNA-binding modules, each recognizing a specific base pair of DNA. By appropriately designing these DNA-binding modules, it is possible to specifically cleave the target base sequence of the ovomucoid locus.

[0050] The CRISPR-Cas system uses a guide RNA with a nucleotide sequence complementary to the target nucleotide sequence adjacent to the PAM sequence on the genome, along with the Cas9 nuclease. The guide RNA includes a CRISPR RNA (crRNA) complementary to the target nucleotide sequence and an auxiliary tracrRNA. The Cas9 nuclease, upon recognizing the guide RNA bound to the target nucleotide sequence at the ovomucoid locus, cleaves the double-stranded DNA in the region consisting of the target nucleotide sequence 5' end to the PAM sequence.

[0051] Breakage of double-stranded DNA by programmable endonucleases is extremely rapid in cells because it can lead to loss of a large amount of genetic information or cancer. One of the main repair pathways, non-homologous end join repair, which joins the broken ends together, introduces mutations (deletions or insertions) into the genome's base sequence with a high probability. Therefore, when using TALENs, the TALEN should be designed according to the base sequences (effector sequences) of regions located at the 5' and 3' ends of the site to be modified in the ovomucoid locus, respectively, and recognized by the DNA binding module. Suitable effector sequences for the site to be modified in the ovomucoid locus can be identified, for example, using "TALEN Targeter" (https: / / tale-nt.cac.cornell.edu / ). Alternatively, when using the CRISPR-Cas system, the target base sequence in the ovomucoid gene can be identified using "CRISPR direct" (http: / / crispr.dbcls.jp / ), for example.

[0052] In the modification step, for example, a vector expressing a programmable endonuclease can be introduced into the epiSC by known methods such as microinjection, electroporation, calcium phosphate, and lipofection. For example, when introducing a vector expressing TALEN as a programmable endonuclease into the epiSC, a vector expressing TALEN designed according to the target base sequence of each strand of the genomic DNA double helix is ​​used. When using the CRISPR-Cas system as the programmable endonuclease, a vector expressing guide RNA and Cas9 nuclease can be similarly introduced into the epiSC.

[0053] Modification of the ovomucoid locus involves introducing a mutation at any site in the ovomucoid locus, preferably at least one exon of exons 1-3 or a signal sequence, so that ovomucoid is not expressed. Preferably, the mutation causes at least one exon of exons 1-3 to contain a stop codon, or exon 1 to not contain a start codon. For this purpose, a programmable endonuclease can be designed to cleave at least one exon of exons 1-3 or a signal sequence. The programmable endonuclease is designed by known methods depending on the nucleotide sequence near the cleavage site.

[0054] Specifically, TALEN consists of TALEN left (the first nuclease) and TALEN right (the second nuclease). When cleaving exon 1, for example, TALEN left and TALEN right consist of the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively. When cleaving exon 3, for example, TALEN left (the third nuclease) and TALEN right (the fourth nuclease) consist of the amino acid sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0055] Furthermore, TALEN left may consist of an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6, as long as it has nuclease activity that specifically cleaves the target base sequence. Similarly, TALEN right may consist of an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7, as long as it has nuclease activity that specifically cleaves the target base sequence.

[0056] 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 consisting of the nucleotide sequence shown in any of 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 consisting of the nucleotide sequence shown in sequence number 12 or sequence number 13 of the ovomucoid locus.

[0057] Preferably, an oligoDNA expressing the guide RNA is used to introduce the guide RNA into the epiSC. The base sequence of the oligoDNA is determined based on the target base sequence. When the Cas9 nuclease cleaves the region consisting of the base sequence shown in SEQ ID NO: 8, the sense base sequence of the oligoDNA expressing the guide RNA in the epiSC includes the base sequence shown in SEQ ID NO: 14, and the antisense base sequence of the oligoDNA includes the base sequence shown in SEQ ID NO: 15. In addition, when the base sequence of the region cleaved by the Cas9 nuclease is the base sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, combinations of base sequences included in the sense and antisense base sequences of the oligoDNA include 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. Furthermore, if the base sequence of the region cleaved by the Cas9 nuclease is the base sequence shown in either SEQ ID NO: 12 or SEQ ID NO: 13, then the combinations of base sequences included in the sense and antisense base sequences of the above-mentioned oligoDNA include SEQ ID NO: 22 and SEQ ID NO: 23, and SEQ ID NO: 24 and SEQ ID NO: 25, respectively.

[0058] Whether or not the ovomucoid locus was modified in the above modification step can be determined by analyzing the base sequence of the ovomucoid locus on the epiSC genome. For example, after introducing a programmable endonuclease, genomic DNA can be recovered from the stably proliferating epiSC and the base sequence of the ovomucoid locus can be analyzed.

[0059] Furthermore, in order to efficiently obtain chimeric individuals with genomes modified to prevent ovomucoid expression, epiSCs in which a knockout mutation preventing ovomucoid gene expression has been introduced at the ovomucoid locus may be selected. In addition, to enrich cells into which a programmable endonuclease vector has been introduced, an expression system that transiently expresses drug resistance genes such as puromycin may be introduced into the epiSCs.

[0060] Next, the transplantation step will be explained in detail. In the transplantation step, an epiSC with a modified ovomucoid locus is transplanted into a bird embryo. The transplantation procedure is not particularly limited, but it is sufficient to inject the epiSC into the bird embryo using a tubule.

[0061] Specifically, in the transplantation step, for example, an epiSC in which the ovomucoid gene locus has been modified so that ovomucoid is not expressed can be transplanted into the blastocyst of a fertilized egg embryo immediately after laying, which has been irradiated with gamma rays. At this time, by using recipient strains with different plumage colors from the epiSC strain, chimeric individuals can be easily identified based on plumage color. For example, when producing chimeric chickens, it is preferable that an epiSC derived from a barred Plymouth Rock breed with black plumage in the chick stage is transplanted into an embryo of a white Leghorn breed with white plumage in the chick stage.

[0062] Following the transplantation step, chimeric individuals can be produced by hatching eggs containing embryos with transplanted epiSCs. The incubation period is approximately 20 days in chickens. In chimeric individuals, sperm and eggs are formed that contain a genome with a modified ovomucoid locus. Therefore, by mating chimeric individuals, genetically modified birds that inherit a genome homozygous for the modified ovomucoid locus are produced with a high probability. Because the ovomucoid locus in these genetically modified birds is modified, the ovomucoid content in the eggs laid by these genetically modified birds is reduced compared to wild-type birds, or the eggs laid by these genetically modified birds do not contain ovomucoid at all.

[0063] Genetically modified birds can be identified by their plumage color, as mentioned above. They can also be identified by analyzing the base sequence of their genomic DNA using methods such as Southern blotting.

[0064] As described in detail above, in the method for producing birds according to this embodiment, the ovomucoid gene locus of avian pluripotent stem cells is cut and modified with a programmable endonuclease, thereby obtaining birds in which ovomucoid is not normally expressed. Since the genome of such birds is inherited through reproductive genes, the ovomucoid content in the eggs laid by such birds is reduced compared to wild-type birds, or the eggs laid by such birds do not contain ovomucoid at all. This makes it possible to sufficiently reduce the allergenicity of the eggs.

[0065] Furthermore, in the modification step of this embodiment, the ovomucoid gene locus of epiSC may be modified. Since epiSC can be easily established from approximately 60,000 blastocyst cells obtained from a single embryo and can be stably cultured while maintaining pluripotency, the ovomucoid gene locus can be modified more reliably. [Examples]

[0066] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

[0067] Example 1: Preparation of TALEN expression vector (Selection of ovomucoid target sequences) To introduce mutations into the ovomucoid gene using TALEN, effector sequences were searched for in the nucleotide sequences of exons 1, 2, and 3 of the ovomucoid gene using TALEN Targeter. As a result, as shown in the underlined region of Figure 2, one pair of effector sequences was found in each of the nucleotide sequences of exon 1 and exon 3. No effector sequences were found in exon 2.

[0068] (Construction of TALEN expression vectors and investigation of TALEN cleavage activity) First, modules that could be bound to each effector sequence in exon 1 and exon 3 were constructed using the 6-module assembly method, and two Golden Gate TALEN expression vectors (left and right) were prepared for exon 1 and exon 3, respectively. The Golden Gate TALEN expression vectors (hereinafter also simply referred to as "G-TALEN expression vectors") were prepared using the Golden Gate TALEN and TAL Effector Kit 2.0 and the Yamamoto Lab TALEN Accessory Pack (both available from Addgene), following the protocols included with the kits.

[0069] Next, to investigate the cleavage activity of TALENs, a Single-Strand Annealing (SSA) assay was performed using HEK293 cells. In the SSA assay, a reporter vector containing a nucleotide sequence targeted by TALENs and a G-TALEN expression vector were co-introduced into HEK293 cells, and cleavage activity was measured from reporter activity.

[0070] The reporter vector was prepared by inserting an annealed synthetic oligonucleotide into pGL4-SSA, which is included in the Yamamoto Lab TALEN Accessory Pack. First, pGL4-SSA was treated with BsaI, subjected to electrophoresis without dephosphorylation, and then excised. For exon 1, the nucleotide sequences of the sense oligonucleotide and antisense oligonucleotide included in the inserted synthetic oligonucleotide are shown in SEQ ID NO: 26 and SEQ ID NO: 27, respectively. For exon 3, the nucleotide sequences of the sense oligonucleotide and antisense oligonucleotide included in the inserted synthetic oligonucleotide are shown in SEQ ID NO: 28 and SEQ ID NO: 29, respectively.

[0071] The details of the solution for annealing the synthetic oligo are shown below. 1 μl of 10x buffer (400 mM Tris-HCl (pH 8), 200 mM MgCl2, 500 mM NaCl) Sense Oligo (50 μM) 1 μM Antisense oligonucleotide (50 μM) 1 μM Sterile distilled water 7 μM The synthetic oligo was annealed by maintaining the above annealing solution at 95°C for 5 minutes, and then cooling it to 25°C over 90 minutes.

[0072] Next, the annealed synthetic oligonucleotide was inserted into BsaI-treated pGL4-SSA. When the subcloned cells were cultured in small cultures and treated with KpnI, two bands of 3800 bp and 1800 bp appeared, confirming that the synthetic oligonucleotide had been inserted.

[0073] Next, the reporter vector was sequenced using the following procedure. After treating the reporter vector with NarI, it was subjected to electrophoresis, the gel was cut out, and the gel fragments were collected in microcentrifuge tubes. These were placed in a deep freezer for about 10 minutes to freeze completely, then thawed by warming with a finger, and spun down in a centrifuge. Approximately 6-8 μl of the extracted liquid was taken and used as a sequencing template.

[0074] Luc2-up-F (SEQ ID NO: 30) or Luc2-down-R (SEQ ID NO: 31) were used as primers for sequence analysis. After confirming that the correct nucleotide sequence had been inserted, the reporter vector was purified using a transfection-grade Miniprep kit, its concentration was quantified, and it was prepared to 150 ng / μl.

[0075] A DNA solution containing the following four plasmids was transfected into HEK293 cells. G-TALEN expression vector (Left) 200 ng G-TALEN expression vector (Right) 200ng Reporter vector 100ng pRL-CMV (reference vector) 20ng

[0076] HEK293 cells were cultured to 70-80% confluence in a 10 cm diameter petri dish. The required amounts of serum-free Dulbecco's Modified Eagle's Medium (hereinafter referred to as "DMEM") for DNA dilution and serum-free DMEM for Lipofectamine LTX dilution were dispensed into microcentrifuge tubes. 25 μl of serum-free DMEM for DNA dilution was added to each well of a 96-well plate, and 4-8 μl of the DNA solution was added to each well and mixed. For LTX dilution, 0.7 μl of LTX was added to the serum-free DMEM so that it was suspended in a well (25 μl), and 25 μl was quickly added to each well and mixed. This was repeated for the required number of plates. The medium was removed from the cells in the petri dish, and 15% fetal bovine serum (hereinafter referred to as "FBS") / DMEM was added and the cells were suspended by direct pipetting on the petri dish. The cell count was measured using a hemocytometer, and 6 × 10⁶ cells were obtained. 5 Adjusted to the number of cells / ml.

[0077] Thirty minutes after adding LTX to the first well, 100 μl of the prepared cells were added to each well and incubated in a 37°C CO2 incubator. Twenty-four hours after transfection, luciferase activity was measured using a Dual-Glo Luciferase Assay System (Promega) according to the instructions.

[0078] (result) Figure 3 shows the cleavage activity of G-TALEN expression vectors as determined by the SSA assay. The positive control TALEN is a TALEN expression vector that targets HPRT1 and exhibits sufficient cleavage activity. Relative activity is the relative value when the cleavage activity of HPRT1 in HEK293 cells is measured and this value is set to 1. The negative control shows the relative activity value when a G-TALEN expression vector is introduced into HEK293 cells that do not contain the target sequence. As shown in Figure 3, no cleavage activity was observed in G-TALEN expression vectors targeting exon 1, while cleavage activity was observed only in G-TALEN expression vectors targeting exon 3.

[0079] Example 2: Mutation introduction of G-TALEN expression vector into chicken epiSCs and confirmation of mutation introduction by Cel-I assay. A G-TALEN expression vector targeting exon 3, which showed cleavage activity, was used to introduce mutations into chicken epiSCs.

[0080] (Culture of chicken epiSCs) First, blastocyst cells were separated from fresh fertilized eggs immediately after laying using the following procedure. After completely removing the egg white with an egg separator, the fertilized egg was placed in a plastic petri dish so that the blastocyst was positioned above the yolk. A ring of sterile, dried filter paper (made by cutting a 5mm diameter hole in the filter paper and then cutting a circle along the outer edge with scissors) was attached to the fertilized egg so that the blastocyst was positioned in the center. Scissors (small, sharp straight scissors) were inserted along the outer edge of the filter paper ring to cut the blastocyst, along with the vitelline membrane, in a circular shape. Next, the filter paper was slowly lifted diagonally with tweezers to remove as much of the yolk attached to the filter paper ring as possible. At this point, the upper layer of the blastocyst was attached to the filter paper ring.

[0081] Next, the filter paper ring was placed with the yolk side facing upwards and immersed in a petri dish containing sterile PBS (phosphate-buffered saline). The filter paper ring was gently shaken to remove any attached yolk. The filter paper ring was then transferred to a separate petri dish containing sterile PBS and shaken vigorously to separate the blastocyst cells from the filter paper ring into a disc shape. The separated blastocyst cells were collected in a 1.5 ml tube using a micropipette.

[0082] Next, the isolated blastocyst cells were cultured on pre-prepared support cells. Mouse embryonic fibroblast-derived cell lines (STO cells) were used as the support cells. The preparation of the support cells is described below.

[0083] STO cells were seeded in a 10 cm diameter petri dish. The culture medium was 10% FBS (fetal bovine serum)-DMEM (Dulbecco's modified Eagle medium). Culture was carried out under conditions of 5% CO2 and 37°C. The STO cells reached confluence after approximately 3 days of culture and were washed three times with cold PBS. The cells were then detached with 0.025% trypsin, 0.02% EDTA 2Na-PBS and seeded in a 15 cm diameter petri dish. After the cells reached confluence, mitomycin C was added to the culture medium to a final concentration of 10 μg / ml, and the cells were cultured for 2 hours. The cells were washed five times with cold PBS, detached with 0.025% trypsin, 0.02% EDTA 2Na-PBS, and centrifuged at least three times. The cell count was calculated using a hemocytometer.

[0084] For culturing the supporting cells, gelatin-coated culture dishes with a diameter of 6 cm were used. Before use, the gelatin coating solution was prepared by adding gelatin to distilled water to a concentration of 0.1%, then dissolving and sterilizing it in an autoclave. The culture dishes were coated at 37°C at least 2 hours before culturing the supporting cells, ensuring the bottom was submerged in the gelatin coating solution. After removing the gelatin coating solution, the mitomycin C-treated supporting cells were placed in 2-3 × 10⁶ cells per 6 cm diameter culture dish. 5 The cells were prepared in 10% FBS-DMEM and seeded to ensure cell development. Support cells were used within 5 days from the day after seeding.

[0085] (Introducing mutations into chicken epiSCs) Blastodisc cells isolated from a single embryo were cultured in a single culture dish seeded with supporting cells. The composition of the culture medium used for culturing the blastodisc cells is shown in Table 1. The medium was based on KnockOut-DMEM as shown in Table 1, and recombinant chicken leukemia suppressor (recombinant chicken LIF) was added to the minimum required amount of warmed medium immediately before use.

[0086] The preparation method for recombinant chicken LIF is described below. Chicken embryo cell line CHCC-OU2 was cultured at 37°C under 5% CO2 in low-glucose DMEM (Invitrogen) 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 SEQ ID NO: 32 and the reverse primer shown in SEQ ID NO: 33. The PCR products were treated with Nhe I and Sal I and subcloned into a pSecTag2A plasmid (Invitrogen) containing a histidine tag.

[0087] Next, the myc-epitope was removed from the pSecTag2A plasmid using restriction enzymes. The recombinant plasmid was introduced into CHCC-OU2 using Polyfect Transfection Reagent (Qiagen), and cells were selected in a medium containing 0.25 μg / ml zeosin (Invitrogen). Stable cell lines secreting biologically active LIF were selected, and recombinant chicken LIF was purified from the culture supernatant using ProBond resin (Invitrogen).

[0088] [Table 1]

[0089] EpiSCs obtained by culturing blastocyst cells on support cells for 2-3 days were introduced with 6.5 μg of G-TALEN expression vector using FuGENE HD (Promega). 24 hours after introduction of the G-TALEN expression vector, puromycin was added to the culture medium at a concentration of 2 μg / mL and cultured for 48 hours. After 48 hours, the medium was changed to remove puromycin, and the cells were cultured for approximately 10 days until the epiSCs proliferated stably.

[0090] Genomic DNA was recovered from stably amplified epiSCs using the DNeasy Blood & Tissue Kit (QIAGEN), and genomic PCR was performed. The genomic PCR conditions were 35 cycles, with each cycle consisting of 2 minutes at 94°C, 10 seconds at 98°C, 30 seconds at 68°C, and 2 minutes at 72°C. The nucleotide sequences of the forward and reverse primers used in genomic PCR are shown in SEQ ID NOs. 34 and 35, respectively.

[0091] (Cel-I assay) Next, the PCR product was re-hybridized, treated with Surveyor nuclease, and subjected to a Cel-I assay to cleave at the heteroduplex region. The SURVEYOR® Mutation Detection Kit (Transgenomic) was used for the Cel-I assay. The PCR product was purified using the Wizard SV Gel and PCR Clean-up System (Promega). DNA elution was performed using 15 μl, and the DNA concentration was quantified after elution.

[0092] Next, a DNA solution for the Cel-I assay was prepared with the following composition. PCR product 400ng 10×hybridization buffer (100mM Tris-HCl (pH8.5), 750mM KCl, 15mM MgCl2) 0.8μl Prepare 8 μl with sterile distilled water. The above DNA solution was maintained at 95°C for 5 minutes and then cooled to 25°C over 60-90 minutes.

[0093] To the DNA solution, 0.4 μl of Enhancer S and 0.4 μl of Nuclease S were added, thoroughly pipetted, and incubated at 42°C for 30 minutes. Immediately after the reaction, the entire volume was subjected to electrophoresis on an agarose gel or polyacrylamide gel.

[0094] (result) In the Cel-I assay, no clear band indicating mutational introduction was obtained. On the other hand, analysis of the nucleotide sequence of the PCR product revealed two types of mutations in the target region: a single nucleotide addition and a single nucleotide substitution, as shown in Figure 4 (see underlined section). However, neither of these mutations resulted in a stop codon.

[0095] Example 3: Preparation of Platinum Gate TALEN expression vector and evaluation of cleavage activity A Platinum Gate TALEN (hereinafter also simply referred to as "P-TALEN") expression vector, a highly active TALEN targeting the exon 1 nucleotide sequence described above, 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 prepared for 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 protocols provided with the kits. The cleavage activity of the P-TALEN expression vectors was evaluated by the SSA assay as described above.

[0096] (result) Figure 5 shows the cleavage activity of the P-TALEN expression vector as determined by the SSA assay. The P-TALEN expression vector showed superior cleavage activity compared to the G-TALEN expression vector that targeted exon 3, which was prepared in Example 1.

[0097] Example 4: One vectorization of P-TALEN and introduction of mutations into chicken epiSCs To improve the efficiency of P-TALEN expression vector introduction, two types of vectors, a P-TALEN expression vector (Left) and a P-TALEN expression vector (Right), were combined into a single vector. Furthermore, a puromycin resistance gene expression cassette was introduced into the single vector to transiently enrich the cells into which the vector had been introduced. Figure 6 shows the composition of the constructed single vector. The cleavage activity of the constructed single vector was evaluated using the SSA assay as described above. For the SSA assay, chicken embryo fibroblasts (CEFs) were used to evaluate cleavage activity within chicken cells.

[0098] 6.5 μg of one vector was introduced into cultured epiSCs using FuGENE HD (Promega). In addition, to increase the mutagenesis efficiency, chicken exonuclease I expression vector (EXO I) was introduced into the epiSCs along with the one vector, independently of the introduction of the one vector alone. 24 hours after the introduction of the one vector, puromycin was added to the culture medium at a concentration of 2 μg / mL and cultured for 48 hours. After 48 hours, the medium was changed to remove the puromycin, and the epiSCs were cultured for approximately 10 days until they were able to grow stably.

[0099] Genomic DNA was recovered from epiSCs in the same manner as described above, and genomic PCR was performed. The nucleotide sequences of the forward and reverse primers used in genomic PCR are shown in SEQ ID NO: 36 and SEQ ID NO: 37, respectively. Furthermore, a Cel-I assay was performed using the PCR product.

[0100] (result) Figure 7 shows the cleavage activity of the one vector by SSA assay. Relative activity is a relative value with the measured value when the one vector was introduced into a CEF that did not contain the target sequence set to 1. As shown in Figure 7, the one vector was shown to have sufficient cleavage activity. Note that "CMV-ptTALEN L+R" refers to a vector in which the expression of Left and Right TALENs is controlled by the CMV promoter, and "CAG-ptTALEN L+R" refers to a vector in which the expression of Left and Right TALENs is controlled by the CAG promoter. Genomic PCR showed a shift band indicating heteroduplex caused by mutation in the drug-selected system, as shown in Figure 8(A). Cel-I assay showed a band of digested fragments indicating mutation introduction from the genome of drug-selected epiSCs, as shown in Figure 8(B). No effect was observed from the introduction of chicken EXO I.

[0101] Example 5: Introduction of knockout mutations into epiSC and cloning The mutant regions of the epiSC genomic DNA were amplified by PCR, cloned into a vector, and the nucleotide sequences of the mutant regions were analyzed. Analysis of 43 clones revealed 10 deletions, 1 insertion, and 2 substitutions. The mutation efficiency was high at 30%. Analysis of the knockout mutations revealed knockout mutations due to stop codon insertions in 3 clones (2 deletions and 1 insertion). Figure 9 shows representative nucleotide sequences where mutations were introduced, specifically #3 (A) and #36 (C) with deletions, and #4 and #18 (B) with insertions. In Figure 9, the double underline in the wild type (D) indicates the signal sequence, while the underlines in #3, #18, and #36 indicate the amino acid sequences mutated by the mutation. The knockout mutation efficiency was 7%.

[0102] After introducing the one vector into epiSCs, 3300 cells were seeded into 96-well plates and cloned. EpiSC colonies grew from 49 wells across a total of 9 plates, and ultimately, growth was successfully achieved in 27 wells. The cells from these 27 wells were cryopreserved, and genomes were extracted from a portion of each well. The Cel-I assay was performed as described above, and the base sequence was further analyzed.

[0103] (result) Of the 27 wells, 4 were confirmed to be positive for the Cel-I assay. After stably growing each cell, as exemplified by clone #5 in Figure 10, the nucleotide sequence was analyzed. As shown in Figure 11, in the nucleotide sequence of the mutant region of cloned ovomucoid knockout epiSC strain #4, a "T" was inserted after the 35th base from the 5' end. When this nucleotide sequence was converted to an amino acid sequence, it was found that stop codons were inserted at the positions corresponding to the 26th and 31st residues from the N-terminus.

[0104] On the other hand, the nucleotide sequence of the mutant region of ovomucoid knockout epiSC strain #5 showed a deletion of 5 nucleotides at the same positions as the nucleotide sequence shown in Figure 9(C). When this nucleotide sequence was converted to an amino acid sequence, it was found that stop codons were inserted at positions corresponding to the 24th and 29th residues from the N-terminus.

[0105] When the amino acid sequence encoded by the nucleotide sequence of epiSC strain #4 was compared with the amino acid sequence of wild-type ovomucoid, it was found that, as shown in Figure 12(A), a frameshift caused an amino acid mutation starting from the 13th residue from the N-terminus of the signal peptide, and that the signal peptide was translated up to the 25th residue. Since the signal peptide is cleaved in the endoplasmic reticulum of the cell and not secreted, this mutation was confirmed to be a knockout mutation of ovomucoid.

[0106] When the amino acid sequence encoded by the nucleotide sequence of epiSC strain #5 was compared with the amino acid sequence of wild-type ovomucoid, it was found that, as shown in Figure 12(B), a frameshift caused an amino acid mutation starting from the 11th residue from the N-terminus of the signal peptide, and that the mutation was translated up to the 23rd residue within the signal peptide. This confirmed that epiSC strain #5 is also an ovomucoid knockout mutation. Furthermore, although the ovomucoid knockout epiSC strain #5 was confirmed to be completely cloned based on the nucleotide sequence analysis, as a precaution, another cloning was performed, and ovomucoid knockout epiSC strain #5-3 was also prepared.

[0107] Example 6: Creation of Chimera Chickens Ovomucoid knockout epiSC strains #4, #5, and #5-3 were transplanted into the blastocyst of fertilized egg embryos immediately after laying, which had been irradiated with 5 Gy of gamma rays, and germ cell chimeric chickens (G0) were hatched.

[0108] (result) Eighteen chimeric chickens were successfully produced. The ovomucoid knockout epiSC strain is a barred Plymouth Rock breed (black plumage as a chick), and the recipient embryo for transplantation is a white Leghorn breed (white plumage as a chick). Therefore, in the case of a chimera, the ovomucoid knockout epiSC differentiates into the epidermis, resulting in black plumage. Figures 13(A) and (B) show the appearance of chimeric chickens derived from epiSC strain #5 and #4, respectively. Black plumage was observed in these chimeric chickens. As shown in Table 2, the chimeric chickens consisted of 6 males, 7 females, and 5 undetermined. Of the 18 chickens, 11 were black-feathered chimeras. "Plumage" in Table 2 indicates the percentage of black-feathered chickens.

[0109] [Table 2]

[0110] By mating the resulting male and female chimeric chickens, homozygous chickens (G1) with the ovomucoid gene knocked out can be produced. Since the ovomucoid gene is knocked out in these chickens, the eggs they lay do not contain ovomucoid.

[0111] Example 7: Construction of a CRISPR / Cas9 vector To construct the CRISPR / Cas9 vector, the puromycin resistance gene was inserted into the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (Addgene) as shown below, similar to the case of TALEN (see Figure 14).

[0112] First, we used "CRISPR direct" (http: / / crispr.dbcls.jp / ) to search for target sequences that could induce knockout of the ovomucoid gene. As a result of the search, we identified four target sequences in exon 1 and two in exon 2 (SEQ ID NOs. 8-13). Based on these target sequences, we synthesized oligo DNA with the nucleotide sequences shown in Figure 15. In Figure 15, "sense" targets the positive strand of the ovomucoid gene, and "antisense" targets the negative strand. The underlined nucleotide sequences in Figure 15 indicate the additional sequences for incorporation into the vector.

[0113] Synthesized oligoDNA was introduced into vectors to create six ovomucoid knockout CRISPR / Cas9 vectors (CRISPR / Cas9-Pur). Reporter vectors for SSA assays were also prepared based on the target sequences, and their target sequence cleavage activity was evaluated. For exon 1, the nucleotide sequences of the sense and antisense oligos contained in the synthetic oligo inserted into the SSA assay reporter vector are shown in SEQ ID NOs. 38 and 39, respectively. For exon 3, the nucleotide sequences of the sense and antisense oligos contained in the inserted synthetic oligo are shown in SEQ ID NOs. 40 and 41, respectively.

[0114] (Investigation of CRISPR / Cas9 cleavage activity) To measure the target sequence cleavage activity of the ovomucoid knockout CRISPR / Cas9 vector, the CRISPR / Cas9 vector was introduced into HEK293 cells, followed by a SSA assay. A similar SSA assay was also performed using epiSC to test the cleavage activity in chicken cells.

[0115] (result) Two vectors (exon 1 #1 and exon 1 #2) that targeted exon 1 were found to have approximately twice the cleavage activity of the CMV-ptTALEN L+R vector, which showed high activity in the SSA assay, as shown in Figure 16. Vector #1 was found to have cleavage activity equivalent to TALEN even in epiSC, as shown in Figure 17.

[0116] This example demonstrates that the ovomucoid gene locus in chicken epiSCs can be modified using CRISPR / Cas9. Consequently, it is possible to create chickens in which the ovomucoid gene is knocked out using CRISPR / Cas9.

[0117] The embodiments described above are for illustrative purposes only and do not limit the scope of the present invention. That is, the scope of the present invention is defined not by the embodiments, but by the claims. Various modifications made within the scope of the claims and equivalent inventive meaning are considered to be within the scope of the present invention.

[0118] This application is based on Japanese Patent Application No. 2015-168372, filed on 27 August 2015. The entire specification, claims, and drawings of Japanese Patent Application No. 2015-168372 are incorporated herein by reference. [Industrial applicability]

[0119] The present invention is suitable for the production of bird eggs, and in particular for the production of chicken eggs.

Claims

1. A modification step involves cleaving and modifying the ovomucoid gene locus of avian cells possessing pluripotency and germ cell differentiation ability using a transcription activator-like effector nuclease, A transplantation step involves transplanting the cells, which have been modified at the ovomucoid gene locus, into a bird embryo. Includes, The aforementioned transcription activator-like effector nuclease is, A first nuclease comprising the amino acid sequence shown in SEQ ID NO: 4 and a second nuclease comprising the amino acid sequence shown in SEQ ID NO: 5, In the aforementioned modification step, A one vector, in which a vector expressing the first nuclease and a vector expressing the second nuclease are combined into a single vector, is introduced into the cells. Methods for breeding birds.

2. A modification step involves cleaving and modifying the ovomucoid gene locus of avian cells possessing pluripotency and germ cell differentiation ability using a transcription activator-like effector nuclease, A transplantation step involves transplanting the cells, which have been modified at the ovomucoid gene locus, into a bird embryo. Includes, The aforementioned transcription activator-like effector nuclease is, The first nuclease consists of the amino acid sequence shown in SEQ ID NO: 4, and the second nuclease consists of the amino acid sequence shown in SEQ ID NO:

5. Methods for breeding birds.

3. In the aforementioned transplantation step, The cells containing a mutation in the signal sequence of the ovomucoid gene locus are transplanted into the embryo. A method for producing birds according to claim 1 or 2.

4. In the aforementioned transplantation step, The cells containing a stop codon in the third exon from the 5' end of the ovomucoid locus are transplanted into the embryo. A method for producing birds according to any one of claims 1 to 3.

5. The transplantation step includes a hatching step in which an egg containing the transplanted cells is hatched to produce a chimeric individual, A breeding step involves mating the aforementioned chimeric individuals to produce birds having a genome that homozygously possesses the modified ovomucoid locus, A method for producing birds according to any one of claims 1 to 4, further comprising:

6. A method for producing birds according to any one of claims 1 to 5, This includes obtaining eggs laid by birds produced by the aforementioned method for producing birds. Egg manufacturing method.

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