WO2017033625A1 - 鳥類、鳥類の作出方法および鳥類の卵 - Google Patents
鳥類、鳥類の作出方法および鳥類の卵 Download PDFInfo
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- WO2017033625A1 WO2017033625A1 PCT/JP2016/071237 JP2016071237W WO2017033625A1 WO 2017033625 A1 WO2017033625 A1 WO 2017033625A1 JP 2016071237 W JP2016071237 W JP 2016071237W WO 2017033625 A1 WO2017033625 A1 WO 2017033625A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knockout animals
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L15/00—Egg products; Preparation or treatment thereof
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/30—Bird
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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- C12N2510/00—Genetically modified cells
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- C12N2517/00—Cells related to new breeds of animals
- C12N2517/02—Cells from transgenic animals
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
Definitions
- the present invention relates to birds, bird production methods and bird eggs.
- Chicken eggs are the number one food cause of food allergies in Japan.
- Several proteins in chicken eggs are allergens that cause food allergies. Examples of allergens contained in chicken eggs include ovomucoid, ovalbumin, lysozyme and ovotransferrin.
- Non-Patent Document 1 discloses a genetically modified chicken obtained by knocking out an ovalbumin gene with a transcription activator-like effector nuclease (TALEN). According to the genetically modified chicken, it is considered that chicken eggs not containing ovalbumin can be obtained.
- TALEN transcription activator-like effector nuclease
- Ovomucoid is the most allergenic protein in chicken eggs. Since ovomucoid has high physicochemical stability, allergenicity of ovomucoid is maintained even when heated. Therefore, by the method disclosed in Non-Patent Document 1, even if the ovalbumin is not included in the chicken egg or the ovalbumin is deactivated by heating, the allergenicity of the egg is sufficiently reduced. Is hard to say.
- primordial germ cells are used. Since only a small number of primordial germ cells exist in the development process, a technique for culturing primordial germ cells is required. Several primordial germ cell culture methods have been reported so far, but the reports are limited to several research institutes, and they cannot be cultured even if they are cultured according to the reported methods. There is an inconvenience.
- the present invention has been made in view of the above circumstances, and provides a bird and an avian production method capable of sufficiently reducing the allergenicity of an egg, and an avian egg having a sufficiently reduced allergenicity. With the goal.
- the present inventors knocked out the chicken ovomucoid gene using a conventional homologous recombination method.
- the conventional homologous recombination method required a long period of time for culturing the cells, the cells were damaged and the karyotype was abnormal.
- homozygous chickens in which the ovomucoid gene was knocked out were not obtained.
- the egg produced by the chimeric chicken contained ovomucoid derived from the recipient's genome, and the allergenicity of the egg was not sufficiently reduced.
- the birds according to the first aspect of the present invention are: It lays eggs that do not contain artificially introduced foreign genes in the genome and have a lower ovomucoid content than the wild type.
- the birds according to the first aspect of the present invention are The stop codon is included in at least one of the first to third exons counted from the 5 ′ end of the ovomucoid locus, or the start codon is not included in the first exon counted from the 5 ′ end of the ovomucoid locus, It is good as well.
- the birds according to the first aspect of the present invention are as follows: The signal sequence at the ovomucoid locus has been modified, It is good as well.
- the birds according to the first aspect of the present invention are as follows: A chicken, It is good as well.
- the method for producing birds according to the second aspect of the present invention is as follows.
- the programmable endonuclease is a transcription activator-like effector nuclease, It is good as well.
- the transmission activator-like effector nucleus is: A first nuclease and a second nuclease,
- the first nuclease is Consisting of the amino acid sequence shown in SEQ ID NO: 4
- the second nuclease is Consisting of the amino acid sequence shown in SEQ ID NO: 5, It is good as well.
- the transmission activator-like effector nucleus is: A third nuclease and a fourth nuclease,
- the third nuclease is Consisting of the amino acid sequence shown in SEQ ID NO: 6
- the fourth nuclease is Consisting of the amino acid sequence shown in SEQ ID NO: 7, It is good as well.
- the programmable endonuclease is Clustered Regulated Interspaced Short Palindromic Repeat-Cas9 nuclease in the CRISPR associated protein system, It is good as well.
- the Cas9 nuclease is Cleaving double-stranded DNA in a region consisting of the base sequence shown in SEQ ID NO: 8 of the ovomucoid locus, It is good as well.
- the Cas9 nuclease is Cleaving double-stranded DNA in a region consisting of the base sequence shown in SEQ ID NO: 9 of the ovomucoid locus, It is good as well.
- the Cas9 nuclease is Cleaving double-stranded DNA in a region consisting of the base sequence shown in SEQ ID NO: 10 of the ovomucoid locus, It is good as well.
- the Cas9 nuclease is Cleaving double-stranded DNA in a region consisting of the base sequence shown in SEQ ID NO: 11 of the ovomucoid locus, It is good as well.
- the Cas9 nuclease is Cleaving double-stranded DNA in a region consisting of the base sequence shown in SEQ ID NO: 12 of the ovomucoid locus, It is good as well.
- the Cas9 nuclease is Cleaving double-stranded DNA in a region consisting of the base sequence shown in SEQ ID NO: 13 of the ovomucoid locus, It is good as well.
- the bird egg according to the third aspect of the present invention is: An artificially introduced foreign gene is not included in the genome, and the ovomucoid content is lower than that of the wild type.
- the allergenicity of an egg can be sufficiently reduced.
- an avian egg with sufficiently reduced allergenicity can be obtained.
- FIG. FIG. 6 shows the relative cleavage activity of TALEN targeting exon 1 and exon 3. It is a figure which shows the base sequence of the target region in the genome of the blastoderm upper layer pluripotent stem cell which introduce
- FIG. It is a figure which shows the relative cleavage activity of TALEN which targets exon 3, and highly active type TALEN which targets exon 1.
- FIG. It is a figure which shows the structure of a TALEN expression vector. It is a figure which shows the relative cleavage activity of TALEN in a chicken cell. It is a figure which shows the mutation introduction
- A shows the result of genomic PCR (polymerase chain reaction).
- B shows the results of the Cel-I assay.
- a part of the base sequence of the ovomucoid locus of the pluripotent stem cell line derived from the upper blastoderm having the cloned knockout mutation, the amino acid sequence encoded by the base sequence, and the base sequence of the wild-type ovomucoid locus It is a figure which shows a part of amino acid sequence.
- (A) shows the base sequence and amino acid sequence of the blastodermal upper layer-derived pluripotent stem cell line # 4.
- B shows the base sequence and amino acid sequence of the blastodermal upper layer-derived pluripotent stem cell lines # 5 and # 5-3.
- FIG. 1 It is a figure which shows the photograph of the knockout chimera chicken produced from the blastoderm-derived pluripotent stem cell line which has a knockout mutation.
- A shows a chimeric chicken derived from the blastodermal upper layer-derived pluripotent stem cell line # 5.
- (B) shows a chimeric chicken derived from the blastodermal upper layer-derived pluripotent stem cell line # 4.
- Embodiment 1 First, the first embodiment will be described. Birds according to Embodiment 1 lay eggs that do not contain an artificially introduced foreign gene in the genome and whose ovomucoid content is reduced as compared to the wild type.
- the birds are not particularly limited, and examples thereof include chickens, ducks, turkeys, ducks, quails, pheasants, parrots, finch, hawks, ostriches, emus, and cassowaries.
- the bird is a chicken.
- Chicken varieties are not particularly limited, and include, for example, White Leghorn, Brown Leghorn, Barred Rock, London, New Hampshire, Rhode Island, Australia, Minorca, Amorafort, California.
- the avian genome according to this embodiment does not contain an artificially introduced foreign gene.
- artificially introduced foreign gene refers to a gene having a mutation and a gene not originally included in the genome of the bird, which are artificially introduced by a gene recombination technique or the like. Examples of methods for artificially introducing foreign genes into the genome include homologous recombination methods, retroviral vector methods, lentiviral vector methods, and artificial viral vector methods.
- the avian genome does not include foreign genes introduced by these methods.
- Ovomucoid is a thermostable glycoprotein having a molecular weight of about 28,000. Ovomucoid is produced in the secretory cells of the fallopian tube. Ovomucoid is usually found mainly in the egg white of avian eggs. Ovomucoid, for example, accounts for about 11% by weight of protein contained in egg white of chicken eggs. The content of the ovomucoid in the eggs laid by the birds according to the present embodiment is reduced compared to the wild-type eggs of the same kind of birds.
- the wild type refers to the same kind of birds as the above-mentioned birds whose genes have not been artificially modified.
- the content of ovomucoid in the egg can be quantified using a known technique for detecting the target protein. For example, by subjecting a sample prepared from a wild-type egg and an egg white collected from an egg born by the bird according to the present embodiment to a target by immunostaining with an antibody that binds to ovomucoid in Western blotting, The ovomucoid content can be compared based on the density.
- the ovomucoid content is preferably measured by a sandwich ELISA (Enzyme-Linked Immunosorbent Assay).
- the capture antibody and detection antibody used in the sandwich ELISA may be a monoclonal antibody or a polyclonal antibody.
- the capture antibody and the detection antibody include a rabbit anti-ovomucoid antibody and a mouse anti-ovomucoid antibody, respectively.
- the rabbit anti-ovomucoid antibody a polyclonal antibody obtained by collecting antiserum from a rabbit immunized with ovomucoid and purifying by affinity chromatography using ovomucoid may be used.
- a mouse anti-ovomucoid antibody a mouse anti-ovomucoid antibody purified from ascites antibody by establishing a monoclonal antibody-producing hybridoma against ovomucoid by a cell fusion method using mouse spleen cells may be used.
- the detection antibody is not particularly limited, and may be labeled with peroxidase.
- ovomucoid When labeled with peroxidase, ovomucoid can be quantified by color development of TMB (3,3 ', 5,5'-tetramethylbenzidine). The ovomucoid content may be evaluated by the concentration in the sample. By constructing an appropriate sandwich ELISA, ovomucoid can be quantified with a detection limit of about 50 pg / ml.
- Quantifying the content of ovomucoid as described above can confirm that the content of ovomucoid in eggs laid by birds according to the present embodiment is reduced as compared to the wild type of the same kind of birds.
- the content of the ovomucoid in the eggs laid by birds according to the present embodiment is 95% by weight or less, 80% by weight or less, 60% by weight or less, 40% by weight or less of the content of ovomucoid in wild-type eggs of the same kind of birds. % By weight, 20% by weight or less, 10% by weight or less, or 5% by weight or less.
- the egg which the bird which concerns on this Embodiment lays may have an ovomucoid concentration below a detection limit, when ovomucoid is quantified.
- the ovomucoid in the egg white protein of chicken eggs produced by the chicken is 0 to 8% by weight, 0 to 4% by weight, 0 to 3% by weight, 0 to 2% by weight. Alternatively, it may be 0 to 1% by weight.
- the ovomucoid content may be evaluated by the weight of ovomucoid per unit amount of egg white.
- the eggs produced by the birds according to the present embodiment do not contain ovomucoid.
- not containing ovomucoid means not containing ovomucoid contained in the egg white of the wild-type egg.
- eggs that do not contain ovomucoids also include eggs that contain fragments of ovomucoid that are not full length.
- the ovomucoid locus in the avian genome according to the present embodiment has a knockout mutation that does not express ovomucoid or a mutation that cannot express full-length ovomucoid, and thus ovomucoid does not express normally.
- the mutation is optional as long as the ovomucoid is not normally expressed, but specifically, insertion of a stop codon into an exon within the ovomucoid locus is preferable.
- the ovomucoid locus contains 5 exons. If exons 1 to 5 are counted in order from the first exon counting from the 5 'end, the stop codon may be inserted at any position of exons 1 to 5. More preferably, the bird comprises a stop codon in at least one exon of exons 1-3 of the ovomucoid locus. If a stop codon is inserted into exons 1 to 5 in the ovomucoid locus, the ovomucoid is not completely synthesized and the full-length ovomucoid is not expressed.
- a fragment that is part of the ovomucoid may be expressed, but if the antigenic epitope of the ovomucoid fragment is reduced compared to the full-length ovomucoid, allergenicity is reduced. Can do.
- the mutation may be a mutation of the start codon in the ovomucoid locus. If there is a mutation at the start codon in the ovomucoid locus, synthesis of ovomucoid based on mRNA is not performed. For example, the birds do not contain an initiation codon in exon 1 of the ovomucoid locus.
- the avian has a modified signal sequence at the ovomucoid locus.
- the signal sequence at the ovomucoid locus includes a partial base sequence of exon 1 and a partial base sequence of exon 2, and encodes 25 amino acid residues.
- FIG. 1 shows the base sequences of exon 1, exon 2 and exon 3. In FIG. 1, exon bases are shown in capital letters and intron bases in lower case letters.
- the underlined base sequence shown in FIG. 1 is a signal sequence.
- a mutation may be introduced into the start codon ATG, or a mutation that causes a stop codon in the signal sequence may be introduced.
- the stop codon is contained in exon 1 of the ovomucoid locus.
- the base 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.
- the avian does not contain an artificially introduced foreign gene in its genome. For this reason, in the production of the above-mentioned birds in which the ovomucoid locus has been modified, the genome editing technique described in detail below is used instead of the homologous recombination method in which a foreign gene is replaced with a specific gene on the genome. preferable.
- the birds according to the present embodiment do not normally express ovomucoid, and therefore lay eggs with a reduced ovomucoid content than the wild type.
- Ovomucoid is so allergenic that only ovomucoid is examined independently in addition to egg yolk and egg white in the examination of egg allergy. For this reason, an egg with less ovomucoid content than the wild type can sufficiently reduce the allergenicity of the egg.
- the above birds do not contain an artificially introduced foreign gene in the genome.
- the absence of foreign genes can prevent the appearance of unexpected phenotypes and toxicity.
- by not including a foreign gene it can be avoided as much as possible to impair the certainty of reproductive inheritance in the birds.
- ovomucoid since ovomucoid has high physicochemical stability, the allergenicity of ovomucoid is maintained even in processed foods or vaccines containing egg whites of birds that have been heat-treated. Therefore, the egg in which the ovomucoid content according to the present embodiment is reduced as compared with the wild type is also useful as a raw material for various products such as processed foods and vaccines.
- the birds described above may include a stop codon in at least one exon of exons 1 to 3 of the ovomucoid locus. If the exon 3 contains a stop codon, the antigenic epitope of the secreted ovomucoid fragment is less than that of the full-length ovomucoid, so that allergenicity can be reduced.
- birds according to the present embodiment can lay eggs that do not contain ovumcoid fragments at all, as well as full-length ovomucoid.
- exon 1 when exon 1 is not included in exon 1 of the ovomucoid locus, synthesis of ovomucoid based on mRNA is not performed, so that the birds can lay eggs that do not contain ovomucoid.
- the signal sequence at the ovomucoid locus may be modified. Since the signal peptide corresponding to the signal sequence is cleaved within the endoplasmic reticulum of the cell and is not secreted, if the signal sequence contains a stop codon, secretion of the peptide derived from the ovomucoid locus to egg white can be prevented. As a result, allergenicity caused by ovomucoid can be further reduced.
- the birds according to this embodiment are preferably chickens. Since chickens lay eggs with great demand, they can efficiently supply eggs with allergenicity sufficiently reduced. In terms of supplying edible eggs, quail and the like are preferable in addition to chickens.
- the mutation at the ovomucoid locus may be a mutation that causes a frame shift to an exon within the ovomucoid locus.
- an avian egg that does not contain an artificially introduced foreign gene in the genome and has a reduced ovomucoid content than the wild type.
- hen's egg is widely used as a raw material for confectionery, beverages, processed foods, etc., or used for the production of pharmaceuticals such as vaccines. If the hen's egg which concerns on the said embodiment is used, even if an ovomucoid mixes in various products, the allergenic property of an ovomucoid can be reduced. Furthermore, if avian eggs that do not contain ovomucoid are used, the risk that the most allergenic ovomucoid is mixed into various products can be minimized.
- in vitro fertilization techniques have not been established. Further, only one single-cell fertilized egg can be obtained from one bird laying eggs. Furthermore, it is difficult to identify a one-cell fertilized egg present in the fallopian tube. For these reasons, it is difficult to apply genetic modification techniques to avian fertilized eggs. Therefore, pluripotent stem cells having pluripotency or germ cell differentiation ability are used in place of fertilized eggs in order to produce birds with modified genes.
- the method for producing birds according to the present embodiment includes the steps of modifying the ovomucoid locus by modifying and modifying the ovomucoid locus of the pluripotent stem cell of the bird with a programmable endonuclease (programmable endonuclease). Transplanting the pluripotent stem cells into the avian embryo.
- a programmable endonuclease programmable endonuclease
- avian pluripotent stem cells include embryonic stem cells (ES cells) having pluripotency and germ cell differentiation ability.
- An avian ES cell is, for example, an epiblast-derived stem cell (hereinafter also simply referred to as “epiSC”) that can be established from a blastoderm cell isolated from an upper blastoderm layer of a fertilized egg. It is.
- epiSC epiblast-derived stem cell
- the stage X blastoderm of the developmental stage (I to XIV) of Eyal-Giladi and Kochav consists of the upper blastoderm layer.
- Chicken epiSCs can be obtained by culturing blastoderm cells isolated from the upper blastoderm layer on a feeder cell whose cell growth has been stopped by, for example, irradiation or mitomycin C treatment in a known manner.
- Supporting cells include chicken embryo fibroblasts, mouse embryo fibroblasts, mouse embryo fibroblast-derived cell lines, and the like.
- primordial germ cells may be used. Primordial germ cells can be isolated from avian fetal glands by known methods, for example. In the following, a case where epiSC is used in the modification step will be described.
- the ovomucoid locus on the epiSC genome is cleaved at a specific site with a programmable endonuclease.
- Programmable endonucleases are used in so-called genome editing techniques that can specifically modify (delete, replace, insert) a target site in the genome.
- the programmable endonuclease is designed according to the base sequence of the target DNA, and can cleave DNA with an arbitrary base sequence.
- the programmable endonuclease is not particularly limited, but for example, TALEN, zinc fingerer nuclease (ZFN), and CRISPR-Cas (also called Clustered Regularly Interspersed Short Palindromic Repeat and CrispersascP9) It is.
- TALEN and ZFN are polypeptides composed of a DNA binding domain and a DNA cleavage domain. TALEN and ZFN cleave double-stranded DNA by forming a dimer in close proximity to a pair of DNA cleavage domains at the binding site of the DNA binding domain.
- the DNA-binding domain includes a plurality of DNA-binding modules repeatedly, and each DNA-binding module recognizes a specific base pair of DNA. Therefore, by appropriately designing the DNA-binding module, the DNA-binding domain can be used as a target of the ovomucoid locus.
- the base sequence to be cleaved can be specifically cleaved.
- a guide RNA having a base sequence complementary to the target base sequence adjacent to the PAM sequence on the genome and Cas9 nuclease are used.
- the guide RNA includes CRISPR RNA (crRNA) complementary to the target base sequence and auxiliary tracrRNA.
- the Cas9 nuclease that recognizes the guide RNA bound to the target base sequence of the ovomucoid locus cleaves double-stranded DNA in the region consisting of the target base sequence 5 'to the PAM sequence.
- TALEN TALEN is present depending on the base sequence (effector sequence) of the region present on the 5 ′ end side and 3 ′ end side of the site to be modified at the ovomucoid locus and recognized by the DNA binding module. Just design.
- an effector sequence suitable for the site to be modified at the ovomucoid locus can be identified by, for example, “TALEN Targeter” (https://tail-nt.cac.cornell.edu/).
- TALEN Targeter https://tail-nt.cac.cornell.edu/.
- CRISPR direct http://crispr.dbcls.jp/.
- a vector that expresses a programmable endonuclease may be introduced into epiSC by a known method such as a microinjection method, an electroporation method, a calcium phosphate method, or a lipofection method.
- a vector that expresses TALEN as a programmable endonuclease is introduced into epiSC, a vector that expresses TALEN designed according to the target base sequence of each double strand of genomic DNA is used.
- a vector that expresses guide RNA and Cas9 nuclease may be similarly introduced into epiSC.
- a mutation may be introduced at any site in the ovomucoid locus, preferably at least one exon of exons 1 to 3 or a signal sequence so that the ovomucoid is not expressed.
- the mutation may cause at least one exon of exons 1 to 3 to contain a stop codon, or exon 1 to contain no start codon.
- the programmable endonuclease may be designed so that at least one exon or signal sequence of exons 1 to 3 is cleaved.
- the programmable endonuclease is designed by a known method according to the base sequence near the cleavage site.
- TALEN is TALEN left (first nuclease) and TALEN right (second nuclease).
- TALEN left and TALEN right consist of amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively.
- exon 3 is cleaved, for example, TALEN left (third nuclease) and TALEN right (fourth nuclease) are composed of amino acid sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.
- TALEN left has a nuclease activity that specifically cleaves the target base sequence
- one or several amino acids in the amino acid sequence shown in SEQ ID NO: 4 or 6 are deleted, substituted, or It may consist of an added amino acid sequence.
- TALEN right also has a nuclease activity that specifically cleaves the target nucleotide sequence, one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7. It may be composed of a specific amino acid sequence.
- the Cas9 nuclease when exon 1 is cleaved, can cleave double-stranded DNA in a region consisting of the base sequence shown in any of SEQ ID NOs: 8 to 11 of the ovomucoid locus. Good.
- Cas9 nuclease may cleave double-stranded DNA in a region consisting of the base sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13 of the ovomucoid locus.
- oligo DNA that expresses guide RNA is preferably used.
- the base sequence of the oligo DNA is determined based on the target base sequence.
- the sense base sequence of the oligo DNA that expresses the guide RNA in epiSC includes the base sequence shown in SEQ ID NO: 14,
- the antisense base sequence includes the base sequence shown in SEQ ID NO: 15.
- the base sequence of the region cleaved by Cas9 nuclease is the base sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, the base sequences contained in the oligo DNA sense and antisense base sequences Examples of the combinations 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.
- the base sequence of the region cleaved by Cas9 nuclease is the base sequence shown in either SEQ ID NO: 12 or SEQ ID NO: 13
- the combination of the base sequences contained in the sense and antisense base sequences of the oligo DNA is as follows: SEQ ID NO: 22 and SEQ ID NO: 23, and SEQ ID NO: 24 and SEQ ID NO: 25, respectively.
- Whether or not the ovomucoid locus has been modified in the above modification step can be determined by analyzing the nucleotide sequence of the ovomucoid locus on the epiSC genome. For example, after introducing a programmable endonuclease, genomic DNA is recovered from epiSC that stably grows, and the base sequence of the ovomucoid locus may be analyzed.
- epiSC in which a knockout mutation that does not express ovomucoid gene is introduced into the ovomucoid locus may be selected.
- a knockout mutation that does not express ovomucoid gene is introduced into the ovomucoid locus.
- epiSCs with modified ovomucoid loci are transplanted into avian embryos.
- the transplantation operation is not particularly limited, and epiSCs may be injected into avian embryos using tubules.
- epiSC in which the ovomucoid locus has been modified so that ovomucoid is not expressed may be transplanted into the blastoderm of a fertilized egg embryo immediately after ovulation irradiated with gamma rays.
- a chimeric individual can be easily discriminated based on the feather color by using a recipient having a different color from the epiSC line.
- epiSCs derived from black-faced flaky Magnolia Oak Rock species in chicks are transplanted into white-legged white Leghorn species embryos in chicks.
- the chimera individual can be produced by hatching an egg containing an embryo transplanted with epiSC following the transplanting step.
- the hatching period is about 20 days for chickens.
- sperm and ova with a genome containing a modified ovomucoid locus are formed. Therefore, by crossing chimeric individuals, genetically modified birds that inherited a genome having a modified ovomucoid locus in a homozygote are produced with high probability. Because the genetically modified bird has a modified vomucoid locus, the content of ovomucoid in eggs produced by genetically modified birds is reduced from the wild type, or eggs produced by genetically modified birds do not contain ovomucoid .
- genetically modified birds can be identified by the feather color as described above. Genetically modified birds can also be identified by analyzing the base sequence of their genomic DNA by Southern blotting or the like.
- the ovomucoid locus of avian pluripotent stem cells is cleaved with a programmable endonuclease and modified. Obtainable. Since the genome of the bird is reproductively inherited, the ovomucoid content in the egg laid by the bird is reduced from that of the wild type, or the egg born by the bird does not contain ovomucoid. Thereby, the allergenicity of an egg can fully be reduced.
- the epiSC ovomucoid locus may be modified.
- epiSC can be easily established from blastodermal cells obtained from about 60,000 embryos from a single embryo and can be stably cultured while maintaining pluripotency, so that the ovomucoid locus can be more reliably modified. .
- Example 1 Preparation of TALEN expression vector (Selection of target sequence of ovomucoid)
- the effector sequences were searched with TALEN Targeter for the base sequences of exon 1, exon 2 and exon 3 of ovomucoid.
- TALEN Targeter for the base sequences of exon 1, exon 2 and exon 3 of ovomucoid.
- one set of effector sequences was found in the base sequences of exon 1 and exon 3, as shown in the underlined region in FIG. In exon 2, no effector sequence was found.
- TALEN expression vector (Preparation of TALEN expression vector and examination of TALEN cleavage activity)
- a module capable of binding to each effector sequence in exon 1 and exon 3 was constructed by a 6-module assembly method, and two types of Golden Gate TALEN expression vectors (left and right) were prepared in exon 1 and exon 3, respectively.
- the Golden Gate TALEN expression vector (hereinafter also referred to simply as “G-TALEN expression vector”) is available from Golden Gate TALEN and TAL Effector Kit 2.0 and Yamamoto Lab TALEN Accessory Pack (both available from Addgene). It was prepared according to the protocol attached to
- a single-strand annealing (SSA) assay using HEK293 cells was performed.
- SSA single-strand annealing
- a reporter vector having a base sequence targeted for TALEN and a G-TALEN expression vector were co-introduced into HEK293 cells as follows, and the cleavage activity was measured from the reporter activity.
- the reporter vector was prepared by inserting a synthetic oligo annealed to pGL4-SSA contained in Yamamoto Lab TALEN Accessory Pack. First, pGL4-SSA was treated with BsaI, electrophoresed without dephosphorylation, and then excised.
- exon 1 the base sequences of the sense oligo and the antisense oligo contained in the inserted synthetic oligo are shown in SEQ ID NO: 26 and SEQ ID NO: 27, respectively.
- the base sequences of the sense oligo and the antisense oligo contained in the inserted synthetic oligo are shown in SEQ ID NO: 28 and SEQ ID NO: 29, respectively.
- the annealed synthetic oligo was inserted into pGL4-SSA treated with BsaI.
- the subcloned product was small-cultured and treated with KpnI, two bands of 3800 bp and 1800 bp appeared and it was confirmed that the synthetic oligo was inserted.
- the sequence analysis of the reporter vector was performed according to the following procedure.
- the reporter vector was subjected to NarI treatment, followed by electrophoresis, the gel was cut out, and the gel fragment was recovered in a microtube. This was placed in a deep freezer for about 10 minutes, completely frozen, melted by warming with a finger, and spun down with a centrifuge. About 6-8 ⁇ l of the exuded liquid was taken and used as a template for the sequence.
- Luc2-up-F (SEQ ID NO: 30) or Luc2-down-R (SEQ ID NO: 31) was used as a primer for sequence analysis. After confirming that the correct nucleotide sequence was inserted, the reporter vector was purified using a transfection grade Miniprep kit, the concentration was quantified, and the concentration was adjusted to 150 ng / ⁇ l.
- G-TALEN expression vector (Left) 200 ng
- G-TALEN expression vector (Right) 200 ng
- Reporter vector 100ng
- pRL-CMV reference vector
- HEK293 cells were cultured in a culture dish with a diameter of 10 cm to 70-80% confluence.
- 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 each dispensed into microtubes in the required amount.
- 25 ⁇ l of serum-free DMEM for DNA dilution was added to each well of a 96-well plate, and 4 to 8 ⁇ l of the above DNA solution was added to each well and mixed.
- LTX was added to and suspended in serum-free DMEM for dilution of LTX at a concentration of 0.7 ⁇ l per well (25 ⁇ l), and 25 ⁇ l was quickly added to each well and mixed. This was repeated as many times as necessary.
- the medium was removed from the cells in the culture dish, 15% fetal bovine serum (hereinafter referred to as “FBS”) / DMEM was added, and the cells were directly pipetted on the culture dish to suspend the cells. The number of cells was counted with a hemocytometer and adjusted to 6 ⁇ 10 5 cells / ml.
- FBS fetal bovine serum
- luciferase activity was measured using Dual-Glo Luciferase Assay System (Promega) according to the instruction manual.
- FIG. 3 shows the cleavage activity of the G-TALEN expression vector by SSA assay.
- the positive control TALEN is a TALEN expression vector with sufficient cleavage activity made targeting HPRT1.
- the relative activity is a relative value when the cleavage activity of HPRT1 is measured in HEK293 cells and the measured value is 1.
- the negative control shows a relative activity value when the G-TALEN expression vector is introduced into HEK293 cells not containing the target sequence. As shown in FIG. 3, the cleavage activity was not observed in the G-TALEN expression vector targeting exon 1, and the cleavage activity was observed only in the G-TALEN expression vector targeting exon 3.
- Example 2 Mutagenesis of G-TALEN expression vector into chicken epiSC and confirmation of mutagenesis by Cel-I assay G-TALEN expression vector targeting exon 3 in which cleavage activity was observed was introduced into chicken epiSC. Used for.
- blastoderm cells were isolated from fresh fertilized eggs immediately after egg laying by the following procedure. After complete removal of the egg white with the egg separator, the fertilized egg was allowed to stand in a plastic petri dish so that the blastoderm was positioned above the yolk. A sterilized and dried filter paper ring (having a 5 mm diameter hole in the filter paper and cut into a circular shape with scissors along the outer ring) was attached to the fertilized egg so that the blastoderm was located in the center. A pair of scissors (both sharp and sharp) was inserted along the outer edge of the filter paper ring, and the blastoderm was cut into a circle together with the yolk membrane. Subsequently, the filter paper was slowly lifted obliquely with tweezers to remove as much egg yolk as possible on the filter paper ring. At this time, the upper layer of the blastoderm is attached to the filter paper ring.
- a sterilized and dried filter paper ring having a 5 mm diameter hole in the filter paper and cut into a circular shape with scissors along
- the filter paper ring was immersed in a petri dish containing sterile PBS (phosphate buffered saline) with the yolk side up, and the filter paper ring was gently shaken to remove the adhering yolk.
- the filter paper ring was transferred to a petri dish containing sterile PBS prepared separately, and the blastoderm cells were separated from the filter paper ring in a disc shape by shaking a little. The separated blastoderm cells were collected into a 1.5 ml tube with a micropipette.
- a mouse embryo fibroblast-derived cell line (STO cell) was used as a feeder cell.
- STO cell mouse embryo fibroblast-derived cell line
- STO cells were seeded in a culture dish having a diameter of 10 cm.
- the culture solution is 10% FBS (fetal bovine serum) -DMEM (Dulbecco's modified Eagle medium).
- FBS fetal bovine serum
- Dulbecco's modified Eagle medium The culture was performed under conditions of 5% CO 2 and 37 ° C.
- the STO cells reached confluence in about 3 days of culture, washed 3 times with cold PBS, and then detached with 0.025% trypsin and 0.02% EDTA 2Na-PBS. Sowing.
- mitomycin C was added to the culture solution to a final concentration of 10 ⁇ g / ml, and the cells were cultured for 2 hours.
- the cells were washed 5 times with cold PBS, the cells were detached with 0.025% trypsin and 0.02% EDTA 2Na-PBS, and centrifuged at least 3 times.
- the cell number was calculated using a hemocytometer.
- a gelatin dish of a petri dish for culture having a diameter of 6 cm was used.
- the gelatin coating solution was dissolved and sterilized by autoclaving after adding gelatin to distilled water so that the concentration was 0.1%.
- the culture petri dish was coated at 37 ° C. with the bottom surface immersed in the gelatin coating solution at least 2 hours before culturing the feeder cells.
- the above mitomycin C-treated feeder cells were seeded with 10% FBS-DMEM adjusted to 2 to 3 ⁇ 10 5 cells per 6 cm culture dish. .
- the feeder cells were used within 5 days from the next day after seeding.
- the blastoderm cells isolated from one embryo were cultured in one culture dish seeded with feeder cells.
- Table 1 shows the composition of the medium used for culturing blastoderm cells. The medium was based on KnockOut-DMEM in Table 1, and a recombinant chicken leukemia inhibitory factor (recombinant chicken LIF) was added to a warm minimum necessary amount of medium immediately before use.
- recombinant chicken leukemia inhibitory factor recombinant chicken LIF
- the chicken embryo cell line CHCC-OU2 was obtained with low glucose DMEM (Invitrogen) containing 10% FBS (Hyclone; manufactured by Thermo Fisher Scientific), 100 ⁇ g / ml penicillin and 70 ⁇ g / ml streptomycin under 5% CO 2. And cultured at 37 ° C.
- the LIF coding region 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 product was treated with Nhe I and Sal I and subcloned into pSecTag2A plasmid (Invitrogen) containing a histidine tag.
- the myc-epitope was removed from the pSecTag2A plasmid using a restriction enzyme.
- Polyfect Transfection Reagent manufactured by Qiagen
- the recombinant plasmid was introduced into CHCC-OU2, and cells were selected with a medium containing 0.25 ⁇ g / ml Zeocin (manufactured by Invitrogen).
- a stable cell line secreting biologically active LIF was selected, and recombinant chicken LIF was purified from the culture supernatant using ProBond resin (manufactured by Invitrogen).
- G-TALEN expression vector 6.5 ⁇ g of G-TALEN expression vector was introduced into epiSC obtained by culturing blastoderm cells on feeder cells for 2 to 3 days using FuGENE HD (manufactured by Promega). Puromycin was added to the medium at a concentration of 2 ⁇ g / mL 24 hours after the introduction of the G-TALEN expression vector and cultured for 48 hours. After 48 hours, the medium was changed to remove puromycin from the medium, and the cells were cultured for about 10 days until epiSCs grew stably.
- Genomic DNA was collected from epiSCs stably grown using DNeasy Blood & Tissue Kit (manufactured by QIAGEN) and subjected to genomic PCR.
- the conditions for genomic PCR are 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 base sequences of the forward primer and reverse primer used for genomic PCR are shown in SEQ ID NO: 34 and SEQ ID NO: 35, respectively.
- Cel-I assay a Cel-I assay was performed in which the PCR product was re-hybridized, treated with surveyor nuclease, and cleaved at the heteroduplex portion.
- SURVEYOR trademark
- Mutation Detection Kit manufactured by Transgenomic
- the PCR product was purified using the Wizard SV Gel and PCR Clean-up System (Promega). Elution of DNA was performed in 15 ⁇ l, and after elution, the DNA concentration was quantified.
- a DNA solution for Cel-I assay was prepared with the following composition.
- the DNA solution was maintained at 95 ° C. for 5 minutes and cooled to 25 ° C. over 60-90 minutes.
- Enhancer S 0.4 ⁇ l of Enhancer S and 0.4 ⁇ l of Nuclease S were added, pipetted well, and incubated at 42 ° C. for 30 minutes. Immediately after the reaction, the entire amount was electrophoresed on an agarose gel or polyacrylamide gel.
- Example 3 Preparation of Platinum Gate TALEN Expression Vector and Evaluation of Cleavage Activity Platinum Gate TALEN (hereinafter also simply referred to as “P-TALEN”) expression vector, which is a highly active TALEN targeting the base sequence of exon 1 above was made.
- a module capable of binding to the effector sequence in exon 1 was constructed by a 6-module assembly method, and P-TALEN expression vectors (left and right) were prepared for exon 1.
- a P-TALEN expression vector was prepared using Platinum Gate TALEN Kit and Yamamoto Lab TALEN Accessory Pack (both available from Addgene) according to the protocol attached to the kit.
- the cleavage activity of the P-TALEN expression vector was evaluated by SSA assay as described above.
- FIG. 5 shows the cleavage activity of the P-TALEN expression vector by SSA assay.
- a cleavage activity higher than that of the G-TALEN expression vector targeting exon 3 prepared in Example 1 was observed.
- Example 4 Conversion of P-TALEN into one vector and introduction of mutation into chicken epiSC
- two of P-TALEN expression vector (Left) and P-TALEN expression vector (Right) were used.
- a seed vector was converted into a one vector, and a puromycin resistance gene expression cassette was introduced into the one vector in order to transiently concentrate cells into which the vector had been introduced.
- FIG. 6 shows the construction of the constructed one vector.
- the cleavage activity of the constructed one vector was evaluated by the SSA assay as described above.
- chicken embryo fibroblasts (CEF) were used to evaluate the cleavage activity in chicken cells.
- 6.5 ⁇ g of one vector was introduced into cultured epiSC using FuGENE HD (manufactured by Promega). Independent of the introduction of only the one vector, a chicken exonclease I expression vector (EXO I) was introduced into epiSC together with the one vector in order to increase the mutation introduction efficiency. Puromycin was added to the medium at a concentration of 2 ⁇ g / mL 24 hours after the introduction of the one vector and cultured for 48 hours. After 48 hours, the medium was changed to remove puromycin from the medium, and the cells were cultured for about 10 days until epiSCs grew stably.
- FuGENE HD manufactured by Promega
- EXO I chicken exonclease I expression vector
- Puromycin was added to the medium at a concentration of 2 ⁇ g / mL 24 hours after the introduction of the one vector and cultured for 48 hours. After 48 hours, the medium was changed to remove puromycin from the medium, and the cells were cultured for about 10 days until epiSCs grew
- genomic DNA was collected from epiSC and subjected to genomic PCR.
- the base sequences of the forward primer and reverse primer used for genomic PCR are shown in SEQ ID NO: 36 and SEQ ID NO: 37, respectively.
- Cel-I assay was performed using the PCR product.
- FIG. 7 shows the cleavage activity of the one vector by SSA assay.
- the relative activity is a relative value when the measured value when the one vector is introduced into CEF not containing the target sequence is 1.
- CMV-ptTALEN L + R indicates a vector in which the expression of Left and Right TALEN is controlled by the CMV promoter
- CAG-ptTALEN L + R controls the expression of Left and Right TALEN by the CAG promoter.
- Vector In genomic PCR, as shown in FIG. 8A, a shift band indicating a heteroduplex caused by mutation was observed in the system of drug selection.
- the Cel-I assay as shown in FIG. 8B, a band of a digested fragment indicating mutation introduction was observed from the epiSC genome subjected to drug selection. The effect of introducing chicken EXO I was not observed.
- Example 5 Introduction and cloning of knockout mutation into epiSC
- the mutagenesis region of the epiSC genomic DNA was amplified by PCR, and after cloning into a vector, the nucleotide sequence of the mutagenesis region was analyzed. As a result of analyzing 43 clones, 10 clones were deleted, 1 clone was inserted, and 2 clones were replaced. The mutagenesis efficiency was as high as 30%. When knockout mutations were analyzed, knockout mutations due to insertion of stop codons were detected in 3 clones (2 clones for deletion and 1 clone for insertion).
- Example 6 Production of chimeric chickens Ovomucoid knockout epiSC strains # 4, # 5 and # 5-3 were transplanted into the blastoderm of a fertilized egg embryo immediately after ovulation irradiated with gamma rays at 5 Gy, and a germ cell chimeric chicken (G0 ) was hatched.
- FIGS. 13 (A) and (B) show the appearance of a chimeric chicken derived from epiSC strain # 5 and a chimeric chicken derived from # 4, respectively. These chimera chickens were found to have black feathers. As shown in Table 2, the breakdown of the chimera chicken was 6 males, 7 females, and 5 undecided. Eleven of the 18 birds were black feather chimeras. “Pattern” in Table 2 indicates the ratio of black pouches.
- a homozygous chicken (G1) in which the ovomucoid gene is knocked out can be produced. Since the ovomucoid gene of the chicken has been knocked out, the egg produced by the chicken does not contain ovomucoid.
- Example 7 Construction of CRISPR / Cas9 vector
- puromycin was added to the pX330-U6-Chmericic_BB-CBh-hSpCas9 vector (manufactured by Addgene) as follows. A resistance gene was inserted (see FIG. 14).
- a target sequence capable of inducing knockout of the ovomucoid gene was searched.
- 4 target sequences were determined in exon 1 and 2 in exon 2 (SEQ ID NOs: 8 to 13).
- oligo DNAs having the base sequence shown in FIG. 15 were synthesized.
- “sense” targets the plus strand of the ovomucoid gene
- “antisense” targets the minus strand.
- the underlined base sequence in FIG. 15 indicates an additional sequence for incorporation into a vector.
- the synthesized oligo DNA was introduced into a vector to prepare 6 types of CRISPR / Cas9 vectors for ovomucoid knockout (CRISPR / Cas9-Pur). Based on the target sequence, a reporter vector for SSA assay was also prepared in the same manner as described above, and the cleavage activity of the target sequence was evaluated.
- CRISPR / Cas9-Pur CRISPR / Cas9-Pur
- a reporter vector for SSA assay was also prepared in the same manner as described above, and the cleavage activity of the target sequence was evaluated.
- exon 1 the base sequences of the sense oligo and the antisense oligo contained in the synthetic oligo inserted into the reporter vector for SSA assay are shown in SEQ ID NO: 38 and SEQ ID NO: 39, respectively.
- SEQ ID NO: 40 and SEQ ID NO: 41 SEQ ID NO: 40 and SEQ ID NO: 41, respectively.
- the two vectors (exon 1 # 1 and exon 1 # 2) prepared by targeting exon 1 have about 2 times higher cleavage activity than the CMV-ptTALEN L + R vector showing high activity in the SSA assay. It was found to have As shown in FIG. 17, the vector of # 1 was found to have a cleavage activity equivalent to that of TALEN even in epiSC.
- This example shows that the ovomucoid locus of chicken epiSC can be modified using CRISPR / Cas9.
- CRISPR / Cas9 a chicken in which the ovomucoid gene is knocked out can be produced.
- the present invention is suitable for the production of avian eggs, particularly for the production of chicken eggs.
Abstract
Description
本発明の第1の観点に係る鳥類は、
人為的に導入された外来遺伝子をゲノムに含まない、かつ
オボムコイドの含有量が野生型よりも低減された卵を産む。
オボムコイド遺伝子座の5’末端から数えて1~3番目の少なくとも1つのエクソンに終止コドンを含む、または
オボムコイド遺伝子座の5’末端から数えて1番目のエクソンに開始コドンを含まない、
こととしてもよい。
オボムコイド遺伝子座におけるシグナル配列が改変されている、
こととしてもよい。
ニワトリである、
こととしてもよい。
鳥類の多能性幹細胞のオボムコイド遺伝子座を、プログラマブルエンドヌクレアーゼで切断し、改変する改変ステップと、
オボムコイド遺伝子座を改変した前記多能性幹細胞を、鳥類の胚に移植する移植ステップと、
を含む。
transcription activator-like effector nucleaseである、
こととしてもよい。
第1のヌクレアーゼおよび第2のヌクレアーゼであって、
前記第1のヌクレアーゼは、
配列番号4に示されるアミノ酸配列からなり、
前記第2のヌクレアーゼは、
配列番号5に示されるアミノ酸配列からなる、
こととしてもよい。
第3のヌクレアーゼおよび第4のヌクレアーゼであって、
前記第3のヌクレアーゼは、
配列番号6に示されるアミノ酸配列からなり、
前記第4のヌクレアーゼは、
配列番号7に示されるアミノ酸配列からなる、
こととしてもよい。
Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated proteinシステムにおけるCas9ヌクレアーゼである、
こととしてもよい。
前記オボムコイド遺伝子座の配列番号8に示される塩基配列からなる領域において二本鎖DNAを切断する、
こととしてもよい。
前記オボムコイド遺伝子座の配列番号9に示される塩基配列からなる領域において二本鎖DNAを切断する、
こととしてもよい。
前記オボムコイド遺伝子座の配列番号10に示される塩基配列からなる領域において二本鎖DNAを切断する、
こととしてもよい。
前記オボムコイド遺伝子座の配列番号11に示される塩基配列からなる領域において二本鎖DNAを切断する、
こととしてもよい。
前記オボムコイド遺伝子座の配列番号12に示される塩基配列からなる領域において二本鎖DNAを切断する、
こととしてもよい。
前記オボムコイド遺伝子座の配列番号13に示される塩基配列からなる領域において二本鎖DNAを切断する、
こととしてもよい。
胚盤葉上層由来多能性幹細胞のオボムコイド遺伝子座を改変する、
こととしてもよい。
人為的に導入された外来遺伝子をゲノムに含まない、かつ
オボムコイドの含有量が野生型よりも低減されている。
まず、実施の形態1について説明する。実施の形態1に係る鳥類は、人為的に導入された外来遺伝子をゲノムに含まない、かつオボムコイドの含有量が野生型よりも低減された卵を産む。
次に、実施の形態2について説明する。実施の形態2では、上記実施の形態1に係る鳥類に好適な鳥類の作出方法について説明する。
(オボムコイドの標的配列の選定)
TALENでオボムコイド遺伝子に変異を導入するために、オボムコイドのエクソン1、エクソン2およびエクソン3の塩基配列を対象に、TALEN Targeterでエフェクター配列を検索した。その結果、図2の下線を付した領域に示されるように、エクソン1およびエクソン3の塩基配列においてそれぞれ1組のエフェクター配列を見出した。なお、エクソン2には、エフェクター配列が見出されなかった。
まず、エクソン1およびエクソン3における各エフェクター配列に結合できるモジュールの構築を、6-モジュールアセンブリ法で行い、エクソン1とエクソン3でそれぞれGolden Gate TALEN発現ベクター2種(leftとright)を作製した。Golden Gate TALEN発現ベクター(以下、単に「G-TALEN発現ベクター」ともいう)は、Golden Gate TALEN and TAL Effector Kit 2.0およびYamamoto Lab TALEN Accessory Pack(共にAddgene社より入手可能)を用いて、キットに添付のプロトコールに従って作製した。
10×バッファー 1μl(400mM Tris-HCL(pH8)、200mM MgCl2、500mM NaCl)
センスオリゴ(50μM) 1μM
アンチセンスオリゴ(50μM) 1μM
滅菌蒸留水 7μM
上記アニーリングの溶液を、95℃で5分間維持した後、90分間かけて25℃まで冷却することで、合成オリゴをアニールした。
G-TALEN発現ベクター(Left) 200ng
G-TALEN発現ベクター(Right) 200ng
レポーターベクター 100ng
pRL-CMV(リファレンスベクター) 20ng
図3は、SSAアッセイによるG-TALEN発現ベクターの切断活性を示す。陽性対照TALENは、HPRT1を標的に作製された十分に切断活性を有するTALEN発現ベクターである。相対活性は、HEK293細胞でHPRT1の切断活性を測定し、この測定値を1としたときの相対値である。陰性対照は、標的配列を含まないHEK293細胞にG-TALEN発現ベクターを導入した場合の相対活性値を示す。図3に示すように、エクソン1を標的としたG-TALEN発現ベクターには切断活性は認められず、エクソン3を標的としたG-TALEN発現ベクターでのみ切断活性が認められた。
切断活性が認められたエクソン3を標的としたG-TALEN発現ベクターを、ニワトリepiSCに対する変異導入に使用した。
まず、産卵直後の新鮮な受精卵から次の手順で胚盤葉細胞を分離した。エッグセパレーターで卵白を完全に除去したのち、プラスチック製シャーレ中に胚盤葉が卵黄の上部に位置するように受精卵を静置した。滅菌乾燥させたろ紙のリング(ろ紙に直径5mmの穴を開け、その外輪に沿ってハサミで円形にカットしたもの)を、中央に胚盤葉が位置するように受精卵に張りつけた。ろ紙のリングの外縁に沿ってハサミ(小直剪刀両鋭)を入れ、卵黄膜ごと円形に胚盤葉をカットした。続いて、ろ紙をピンセットでゆっくり斜めに持ち上げて、ろ紙のリングに付着する卵黄を可能な限り除去した。このとき、ろ紙のリングには、胚盤葉上層が張り付いた状態となる。
ひとつの胚から分離した胚盤葉細胞を、支持細胞を播種した1個の培養用シャーレで培養した。胚盤葉細胞の培養に使用する培地の組成を表1に示す。なお、当該培地は、表1のKnockOut-DMEMを基礎とし、組み換えニワトリ白血病抑制因子(recombinant chicken LIF)は、加温した最低限必要な量の培地に使用直前に添加した。
続いて、PCR産物を再ハイブリダイゼーションし、surveyor nucleaseにより処理し、ヘテロデュープレックス部分で切断するCel-Iアッセイを行った。Cel-Iアッセイには、SURVEYOR(商標) Mutation Detection Kit(Transgenomic社製)を用いた。PCR産物をWizard SV Gel and PCR Clean-up System(Promega社製)を用いて精製した。DNAの溶出は15μlで行い、溶出後、DNA濃度を定量した。
PCR産物 400ng
10×hybridization buffer(100mM Tris-HCl (pH8.5)、750mM KCl、15mM MgCl2) 0.8μl
滅菌蒸留水で8μlに調製
上記DNA溶液を、95℃で5分間維持し、60~90分間かけて25℃に冷却した。
Cel-Iアッセイでは、変異導入を示す明瞭なバンドは得られなかった。一方、PCR産物の塩基配列を解析したところ、図4に示すように、標的領域に1塩基付加と1塩基置換の2種の変異が認められた(下線参照)。しかし、これらの変異は、いずれも終止コドンを生じる変異ではなかった。
上記エクソン1の塩基配列を標的とする高活性型のTALENであるPlatinum Gate TALEN(以下、単に「P-TALEN」ともいう)発現ベクターを作製した。上記エクソン1におけるエフェクター配列に結合できるモジュールの構築を、6-モジュールアセンブリ法で行い、エクソン1に対してP-TALEN発現ベクター(leftとright)を作製した。P-TALEN発現ベクターは、Platinum Gate TALEN KitおよびYamamoto Lab TALEN Accessory Pack(共にAddgene社より入手可能)を用いて、キットに添付のプロトコールに従って作製した。P-TALEN発現ベクターの切断活性を上述と同様にSSAアッセイで評価した。
図5は、SSAアッセイによるP-TALEN発現ベクターの切断活性を示す。P-TALEN発現ベクターにおいて、実施例1で作製したエクソン3を標的としたG-TALEN発現ベクターを上回る切断活性が認められた。
P-TALEN発現ベクターの導入効率を向上させるため、P-TALEN発現ベクター(Left)とP-TALEN発現ベクター(Right)の2種のベクターをoneベクター化するとともに、ベクターを導入した細胞を一過性に濃縮するために、ピューロマイシン耐性遺伝子の発現カセットをoneベクターに導入した。図6は、構築したoneベクターの構成を示す。構築したoneベクターの切断活性を、上記と同様にSSAアッセイで評価した。なお、SSAアッセイでは、ニワトリ細胞内での切断活性を評価するために、ニワトリ胚線維芽細胞(CEF)を用いた。
図7は、SSAアッセイによるoneベクターの切断活性を示す。相対活性は、標的配列を含まないCEFにoneベクターを導入した場合の測定値を1としたときの相対値である。図7に示すように、oneベクターが十分な切断活性を有することが示された。なお、「CMV-ptTALEN L+R」は、LeftおよびRightのTALENの発現をCMVプロモーターで制御しているベクターを示し、「CAG-ptTALEN L+R」は、LeftおよびRightのTALENの発現をCAGプロモーターで制御しているベクターを示す。ゲノミックPCRでは、図8(A)に示したように薬剤選抜した系で変異により生じるヘテロデュープレックスを示すシフトバンドが観察された。Cel-Iアッセイでは、図8(B)に示すように、薬剤選抜を行ったepiSCのゲノムから変異導入を示す消化断片のバンドが観察された。ニワトリEXO Iの導入の効果は、認められなかった。
上記epiSCのゲノムDNAの変異導入領域をPCRにより増幅し、ベクターへクローニング後、変異導入領域の塩基配列を解析した。43クローンを解析した結果、欠失が10クローン、挿入が1クローンおよび置換が2クローンであった。変異導入効率は、30%と高値であった。このうちノックアウト変異を解析したところ、3クローン(欠失が2クローンおよび挿入が1クローン)で、終止コドンの挿入によるノックアウト変異が検出された。図9に、変異が導入された代表的な塩基配列として、欠失があった#3(A)および#36(C)と、挿入があった#4および#18(B)とを例示する。図9では、野生型(D)の二重下線はシグナル配列を示し、#3、#18および#36の下線は変異導入により変異したアミノ酸配列を示す。ノックアウト変異の効率は、7%であった。
27ウェルのうち、4ウェルにおいてCel-Iアッセイの陽性が確認された。図10に例示する#5のクローンのように各細胞を安定に増殖させた後、塩基配列を解析した。図11に示すように、クローン化されたオボムコイドノックアウトepiSC株#4の変異導入領域の塩基配列では、5’末端から数えて35番目の塩基の次に「T」が挿入されていた。この塩基配列をアミノ酸配列に変換したところ、N末端から数えて26残基目および31残基目に対応する位置に終止コドンが入ることがわかった。
オボムコイドノックアウトepiSC株#4、#5および#5-3を、5Gyでガンマ線照射した放卵直後の受精卵胚の胚盤葉へ移植し、生殖細胞キメラニワトリ(G0)を孵化させた。
18羽のキメラニワトリを作出できた。オボムコイドノックアウトepiSC株は横斑プリマスロック種(雛で黒羽装)であり、移植用のレシピエント胚は、白色レグホン種(雛で白羽装)であるため、キメラ体であれば、オボムコイドノックアウトepiSCが表皮に分化し、黒の羽装が認められる。図13(A)および(B)は、それぞれepiSC株#5由来のキメラニワトリおよび#4由来のキメラニワトリの外観を示す。これらのキメラニワトリには、黒の羽装が認められた。表2に示すように、キメラニワトリの内訳は、雄が6羽、雌が7羽、未定が5羽であった。18羽のうち11羽が黒羽装キメラであった。表2の「羽装」は黒羽装の割合を示している。
CRISPR/Cas9ベクターを構築するために、以下のように、pX330-U6-Chimeric_BB-CBh-hSpCas9ベクター(Addgene社製)に、TALENの場合と同様にピューロマイシン耐性遺伝子を挿入した(図14参照)。
オボムコイドノックアウト用CRISPR/Cas9ベクターの標的配列の切断活性を測定するために、HEK293細胞にCRISPR/Cas9ベクターを導入後、SSAアッセイを行った。また、ニワトリ細胞内での切断活性を試験するために、epiSCを用いて同様のSSAアッセイを行った。
エクソン1を標的に作製した2種のベクター(エクソン1 #1とエクソン1 #2)は、図16に示すように、SSAアッセイで高い活性を示すCMV-ptTALEN L+Rベクターの約2倍高い切断活性を有することがわかった。#1のベクターは、図17に示すように、epiSC中でもTALENと同等の切断活性を有することがわかった。
Claims (17)
- 人為的に導入された外来遺伝子をゲノムに含まない、かつ
オボムコイドの含有量が野生型よりも低減された卵を産む、鳥類。 - オボムコイド遺伝子座の5’末端から数えて1~3番目の少なくとも1つのエクソンに終止コドンを含む、または
オボムコイド遺伝子座の5’末端から数えて1番目のエクソンに開始コドンを含まない、
請求項1に記載の鳥類。 - オボムコイド遺伝子座におけるシグナル配列が改変されている、
請求項1または2記載の鳥類。 - ニワトリである、
請求項1から3のいずれか一項に記載の鳥類。 - 鳥類の多能性幹細胞のオボムコイド遺伝子座を、プログラマブルエンドヌクレアーゼで切断し、改変する改変ステップと、
オボムコイド遺伝子座を改変した前記多能性幹細胞を、鳥類の胚に移植する移植ステップと、
を含む、鳥類の作出方法。 - 前記プログラマブルエンドヌクレアーゼは、
transcription activator-like effector nucleaseである、
請求項5に記載の鳥類の作出方法。 - 前記transcription activator-like effector nucleaseは、
第1のヌクレアーゼおよび第2のヌクレアーゼであって、
前記第1のヌクレアーゼは、
配列番号4に示されるアミノ酸配列からなり、
前記第2のヌクレアーゼは、
配列番号5に示されるアミノ酸配列からなる、
請求項6記載の鳥類の作出方法。 - 前記transcription activator-like effector nucleaseは、
第3のヌクレアーゼおよび第4のヌクレアーゼであって、
前記第3のヌクレアーゼは、
配列番号6に示されるアミノ酸配列からなり、
前記第4のヌクレアーゼは、
配列番号7に示されるアミノ酸配列からなる、
請求項6記載の鳥類の作出方法。 - 前記プログラマブルエンドヌクレアーゼは、
Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated proteinシステムにおけるCas9ヌクレアーゼである、
請求項5に記載の鳥類の作出方法。 - 前記Cas9ヌクレアーゼは、
前記オボムコイド遺伝子座の配列番号8に示される塩基配列からなる領域において二本鎖DNAを切断する、
請求項9に記載の鳥類の作出方法。 - 前記Cas9ヌクレアーゼは、
前記オボムコイド遺伝子座の配列番号9に示される塩基配列からなる領域において二本鎖DNAを切断する、
請求項9に記載の鳥類の作出方法。 - 前記Cas9ヌクレアーゼは、
前記オボムコイド遺伝子座の配列番号10に示される塩基配列からなる領域において二本鎖DNAを切断する、
請求項9に記載の鳥類の作出方法。 - 前記Cas9ヌクレアーゼは、
前記オボムコイド遺伝子座の配列番号11に示される塩基配列からなる領域において二本鎖DNAを切断する、
請求項9に記載の鳥類の作出方法。 - 前記Cas9ヌクレアーゼは、
前記オボムコイド遺伝子座の配列番号12に示される塩基配列からなる領域において二本鎖DNAを切断する、
請求項9に記載の鳥類の作出方法。 - 前記Cas9ヌクレアーゼは、
前記オボムコイド遺伝子座の配列番号13に示される塩基配列からなる領域において二本鎖DNAを切断する、
請求項9に記載の鳥類の作出方法。 - 前記改変ステップでは、
胚盤葉上層由来多能性幹細胞のオボムコイド遺伝子座を改変する、
請求項5から15のいずれか一項に記載の鳥類の作出方法。 - 人為的に導入された外来遺伝子をゲノムに含まない、かつ
オボムコイドの含有量が野生型よりも低減された、鳥類の卵。
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