WO2015163711A1 - 마이오스타틴 유전자를 표적으로 하는 talen 및 이를 이용한 마이오스타틴 유전자가 녹아웃된 동물을 제조하는 방법 - Google Patents

마이오스타틴 유전자를 표적으로 하는 talen 및 이를 이용한 마이오스타틴 유전자가 녹아웃된 동물을 제조하는 방법 Download PDF

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WO2015163711A1
WO2015163711A1 PCT/KR2015/004068 KR2015004068W WO2015163711A1 WO 2015163711 A1 WO2015163711 A1 WO 2015163711A1 KR 2015004068 W KR2015004068 W KR 2015004068W WO 2015163711 A1 WO2015163711 A1 WO 2015163711A1
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myostatin gene
myostatin
gene
sequence
animal
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PCT/KR2015/004068
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French (fr)
Korean (ko)
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윤희준
김석중
김진수
강금단
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윤희준
주식회사 툴젠
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Publication of WO2015163711A1 publication Critical patent/WO2015163711A1/ko

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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • A01K2227/10—Mammal
    • A01K2227/108—Swine

Definitions

  • Myostatin is a protein belonging to the TGF- ⁇ family and is a regulatory factor that inhibits muscle differentiation and growth during muscle differentiation and is also known as growth differentiation factor 8 (GDF-8). .
  • Myostatin is produced primarily from bone cells and circulates through the blood to act on muscle tissue. Therefore, if there is no myostatin gene in animals or inhibits the activity of myostatin, it can be expected to increase muscle.
  • Humans and cattle (Ravi ambadur et al., Genome Res., 7: 910- 915, 2007), sheep and dogs (Dana S. Mosher et al., Plos genet., 3: 779-786, 2007) have identified a myostatin mutation present in nature.
  • muscle phenotype was increased compared to wild-type.
  • mice knocked out the myostatin gene were made, a small amount of muscle was increased when the gene knockout was a heterozygote and a double-muscle when the knockout was a homozygote. Muscle growth was observed (Se-Jin Lee et al. Nature, 16; 98: 9306-9311, 1997).
  • the present inventors have tried to find a method for producing a transgenic pig knocked out of the myostatin gene. As a result, the present inventors have completed the present invention by confirming that, when using TALEN and somatic cell nuclear transfer technology specific to myostatin gene, it is possible to effectively make a transgenic pig knocked out of myostatin gene.
  • Another object of the present invention is to provide a recombinant expression vector for myostatin gene knockout using TALEN.
  • Another object of the present invention to provide a method for producing a cell knocked out myostatin gene using TALEN and to provide a cell knocked out myostatin gene prepared using the same. Another object of the present invention to provide a method for producing a transgenic animal other than a human knocked out myostatin gene using TALEN and a transgenic animal other than a human knocked out myostatin gene prepared using the same There is.
  • a fusion protein having the activity of binding to and cleaving a specific sequence of a myostatin (MSTN) gene:
  • statin Maio in the present invention is known as GDF8 (. Growth differentiation factor 8), is known as a gene that suppresses muscle growth and differentiation ol.
  • Myostatin is a regulatory ligand belonging to the TGF- ⁇ superfamily and its gene structure is similar to other genes belonging to the TGF- ⁇ superfamily.
  • Myostatin gene sequence and the like can be obtained from a known database (GenBank et al.).
  • Myostatin acts as a negative regulator involved in the development, growth, and regulation of muscle homeostasis in the musculoskeletal structure, so if the animal lacks the myostatin gene or inhibits the activity of myostatin, muscle can be expected to increase.
  • Myostatin mutations in nature have been identified in cattle, sheep and dogs, and the deletion of the myostatin gene showed an increased phenotype compared to the wild-type.
  • mice knocked out the myostatin gene were made, a small amount of muscle was increased when the gene knockout was a heterozygote, and a double-muscle when the knockout was a homozygote. It was observed that the muscles are greatly increased.
  • the myostatin gene can be effectively knocked out in an animal that supplies meat to humans, the production of an animal with increased muscles can be expected. For example, to produce pigs with increased muscle mass, they tried to knock out the myostatin gene in pigs.
  • the term "having the activity of binding to and cleaving a specific sequence of myostatin gene” means that the specific nucleic acid sequence of the myostatin gene is specifically recognized and cleaved. Recognizing a specific nucleic acid sequence of the ostatin specifically, it means that the cleavage is generated in the myostatin gene by the nucleotide cleavage domain connected by the TALE domain and the peptide bond.
  • sequence means a nucleotide sequence regardless of its length, which may be linear, circular or branched DNA or RNA, and may be single helical or double helical.
  • TALE domains can be designed for all specific sequences, but experimentally, the length of specific sequences to which TALE domains bind, the length of the spacer between two half-digits forming one target region, etc. This is known to affect the effective action of TALENs.
  • the half-digits are 15-20 bp in length
  • the spacers are 12-14 bp in length
  • the total sequence length is 42-54 bp
  • the starting sequence of the specific sequence is thymine (T) and the last sequence.
  • A adenine
  • specific positions should be selected so that TALENs can cleave myostatin genes specifically and effectively.
  • four specific sequences of the sequence of the pig myostatin gene using the database of TALEN cleavage ability for various specific sequences possessed by Bioinformatics Co., Ltd. SEQ ID NO: TALEN for 1 to 4 sequences
  • TALE Transcription Activator Like Effector
  • TALE domains of the invention refer to protein domains that bind to nucleotides in a sequence-specific manner by a combination of one or more TALE-repeat parents.
  • the TALE domain includes, but is not limited to, at least one TALE-repeats moiety, specifically 1-30 TALE-repeats moieties.
  • TALE-repeat parents may have the following general amino acid sequence as a site that recognizes a particular nucleotide sequence in the TALE domain:
  • XX refers to the super-modified amino acids at positions 12 and 13 to determine the specificity of base recognition. Specifically, the twelfth and thirteenth amino acids of the TALE-repeat parent recognize one specific nucleic acid.
  • the TALE-repeat modalities recognize C, T when XX is NG, A when XX is NI, and G when XX is NN, respectively, thus combining various TALE-repeat mods
  • a TALE domain capable of recognizing a specific position in the target gene can be prepared.
  • a method for preparing a specific TALE domain is described in detail in the Korean patent application 'Genome Engineering 1 through Designed TAL Effector Nuclease (Application No. 10-2013-7018743).
  • TALEN generally acts as a dimer, it is necessary to design two TALEN monomers to cut a specific position in the target gene (FIG. 1).
  • the specific position on the left to which the TALE domain binds is named as a first half-site and the specific position on the right as a second half-site based on the cleavage site.
  • Two TALEN monomers are each made up of two half-sites in different target genes.
  • One recognizes and the two half-digits are designed to be separated from each other by spacers of 9 to 14-bp. According to a specific example of the present invention, it is separated from each other by a 12-bp spacer and recognizes 21 bp (first half-digit, sequence listing first sequence) and 20 bp (second half-site, sequence listing second sequence), respectively.
  • the TALEN monomer was designed and used to effectively induce knockout of porcine myostatin gene.
  • cutting refers to unlinking the covalently bonded backbone of the myostatin nucleotide molecule, and the "nucleotide cleavage domain" 'is a polypeptide having enzymatic activity for nucleotide cutting of such myostatin. Refers to the sequence.
  • Nucleotide cleavage domains can be obtained from endonucleases or exonucleases. Examples of endonucleases from which nucleotide cleavage domains can be obtained include, but are not limited to, endogenous nucleases, recurrent endonucleases, and the like. It is not limited. Such enzymes can be used as the origin of the nucleotide cleavage domain. In addition, the nucleotide cleavage domain can cleave a single nucleotide sequence, as well as cleave a nucleotide sequence of a double bond, depending on the origin of the cleavage domain.
  • the restriction endonuclease is capable of sequence specific binding with DNA (recognition site), and can cleave DNA in the vicinity of the binding site.
  • Fokl a type lis enzyme, promotes double helix cleavage of DNA at 9 nucleotides from the recognition site in one helix and 13 nucleotides from the recognition site in the other helix.
  • Nucleotide cleavage domains of the invention may be derived from type lis restriction endonucleases, wherein the type lis restriction endonucleases are described, for example, in Fokl, Aarl, Acelll, Acil, AloI, ' BaeI, Bbr7I, Cdil, CjePI, Ecil, Esp3I, Finl, Mbol, sa I or SspD51 may be, but is not limited thereto.
  • the nucleotide cleavage domain of the present invention may be Fokl.
  • the fusion protein of the present invention may further comprise a nuclear localization signal sequence.
  • the nuclear position signal of the present invention TALENs targeting myostatin can be used as a means to move into the nucleus to knock out the myostatin gene.
  • a nucleotide sequence encoding the fusion protein and (b) provides a recombinant expression vector for knockout myostatin gene comprising a transcriptional regulatory sequence operably linked to the nucleotide sequence (operat i ve lyli nked).
  • the expression vector may be introduced into a cell to express a fusion protein (TALEN) of the present invention, and a known expression vector such as a plasmid vector, a cosmid vector, and a bacteriophage vector may be used. It can be easily prepared by those skilled in the art according to any known method.
  • TALEN fusion protein
  • opert i ve ly li nked refers to a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences. Whereby the regulatory sequence controls transcription and / or translation of the other nucleic acid sequence.
  • the transcriptional regulatory sequence bound to the nucleotide sequence encoding the fusion protein is capable of controlling transcription of the nucleotide encoding the fusion protein by operating in an animal cell (eg, a mammalian cell), which is derived from a mammalian virus.
  • Promoters and promoters derived from genomes of mammalian cells such as, for example, cyt omega lo virus (CMV) promoter, adenovirus late promoter, vaccinia virus 7. 5K promoter, SV40 promoter, tk promoter of HSV, RSV Promoter, EF1 alpha promoter, metallothionine promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene and human GM-CSF Including but not limited to promoters of genes.
  • the combinatorial expression vector of the present invention may comprise a polyaninated sequence.
  • a method of producing a cell knocked out myostatin gene comprising the following steps.
  • any method known in the art may be used to introduce a recombinant expression vector for myostatin gene knockout into a cell.
  • a recombinant expression vector for myostatin gene knockout was introduced into porcine fetal fibroblasts using electroporation.
  • the double-strand damage on the myostatin gene can be induced.
  • the cell repairs the damaged area using its own repair mechanism.
  • the cell repairs the damaged region by non-homologous end j oini ng (NHEJ) method, and insertion (i nser ti on) or deletion (de l et i on) at the end of the broken DNA strand is performed. Repairs are made in the direction of error-prone. Thus, mutations are induced in the myostatin gene, which results in knockout of the myostatin gene.
  • the mutation may be a mutation by substitution, deletion, insertion, and a combination thereof.
  • various mutations such as insertion and deletion were induced by the TALEN fusion protein, thereby successfully knocking out the myostatin gene.
  • a cell in which myostatin gene is knocked out by the above method is provided.
  • the cell may be a variety of animal cells, but for the purposes of the present invention, since the myostatin gene may be used in the process of preparing a knocked out transgenic animal, it may specifically mean a cell of an animal to be transformed.
  • a pig fetus for preparing a transformed pig in which the myostatin gene is knocked out Fibroblasts were used to prepare cells knocked out of the myostatin gene.
  • a method for producing a transgenic animal other than a human whose knocked out myostatin gene comprises the following steps:
  • step (c) transplanting the nucleus of the transformed cell of step (a) into a somatic cell nuclear transfer step of transplanting the nucleus of step (b);
  • step (d) implanting the embryo cells in which the somatic cells of step (c) have been nuclear-transplanted into the oviduct of the surrogate mother of the animal to be transformed and pregnant.
  • each step is as follows:
  • the follicles are extracted from the ovary of a female pig and matured in oocyte in vitro mature culture medium (in vi t ro maturat i on medi a, IVM med ia), and then, the oocytes are collected using an egg collecting needle The nucleus of the egg was then removed using chemical and physical methods.
  • step (c) the nucleus of the transformed cell of step (a) is replaced by the nucleus of step (b) Somatic cell nuclear transfer to transplant the removed ovum
  • Somat i c-cel l nuc lear transfer refers to a technique for transplanting a nucleus of a cell into an egg which has already had its nucleus removed. After activating cytin to progress, an individual born from a surrogate mother becomes a genetically identical clone because the genetic material of the donor cell is transferred to the nuclear donor cytoplasm.
  • somatic cell nuclear transfer method cell membrane fusion, cytoplasmic microinjection, or the like can be used.
  • Cell membrane fusion has the advantage of being simple and suitable for large-scale fertilized egg production, and intracellular microinjection has the advantage of maximizing contact between the nucleus and egg material.
  • Fusion of somatic cells and nuclei from which the nucleus has been removed is achieved through a method of fusion by changing the viscosity of the cell membrane through electrical stimulation.
  • a cell membrane fusion method was used in which oocytes from which nuclei were removed and donor cells to be transplanted with the somatic cell nucleus were paralleled with electrodes, and then fusion was performed by applying a DC current pill to the fusion device.
  • step (d) implanting embryos in which the somatic cells of step (C) have been nuclear-transplanted and implanted into a fallopian tube of a surrogate mother of an animal to be transformed
  • Implantation of embryonic cells in the surrogate mother's oviduct can be carried out using a conventional method known in the art, and is not particularly limited.
  • the method for preparing a transgenic animal other than a human in which the myostatin gene is knocked out may further include the following steps.
  • step (e) separating the fetus from the surrogate mother whose pregnancy in step (d) is confirmed and confirming whether the myostatin gene is knocked out;
  • step (g) implanting embryonic cells cultured with the cells transplanted with the somatic cells of step (f) into the oviduct of the surrogate mother to be pregnant.
  • the additional steps above are for more effectively producing a transgenic animal knocked out of the myostatin gene, and the second somatic cell nuclear transfer using the nucleus of the fetal cell whose knockout of the myostatin gene is confirmed. And the embryo is to proceed. Since the specific somatic cell nuclear transfer and embryo transfer method is the same as the above method, the description is omitted to avoid excessive duplication. According to a specific example of the present invention, secondary somatic cell nuclear transfer and embryo transfer could effectively produce a transformed pig in which myostatin gene was knocked out.
  • transgenic animals other than human may be mammalian fish, birds, and the like, and specifically, may be an edible animal which may provide meat useful to humans for the purposes of the present invention.
  • transgenic animals other than human may be mammalian fish, birds, and the like, and specifically, may be an edible animal which may provide meat useful to humans for the purposes of the present invention.
  • a specific example of the invention provides a transformed pig knocked out myostatin gene by the above method.
  • the present invention provides a TALEN having the activity of binding to and cleaving a specific position of the myostatin gene.
  • the present invention provides a recombinant expression vector for myostatin gene knockout using TALEN.
  • the present invention provides a method for producing a transgenic animal other than a human whose myostatin gene is knocked out using TALEN.
  • FIG. 1 is a view showing a general principle of the working TALEN of the present invention.
  • Figure 2 is a chart analyzing and analyzing the activity of TALEN produced by targeting a specific sequence of porcine MSTN gene.
  • A is SEQ ID NO: 1
  • B is SEQ ID NO: 2
  • C is SEQ ID NO: 3
  • D is TALEN for SEQ ID NO: 4 The activity was measured and analyzed.
  • FIG. 3 is a view showing a recombinant expression vector for myostatin gene knockout using TALEN of the present invention.
  • Figure 4 shows the results of sequencing analysis to determine whether the mutation of the MSTN gene in male pig embryos produced through secondary somatic cell nuclear transfer and embryo transfer of the present invention.
  • Bold G of F1 and 18-2, 164bp of 19-3 indicate that the nucleotide sequence is inserted compared to the native form, and "- ⁇ " indicates that the nucleotide sequence is deleted compared to the native form (de l et i on).
  • FIG. 5 is a photograph comparing MSTN ⁇ / ⁇ male pigs and natural pigs produced through secondary somatic cell nuclear transfer and embryo transfer.
  • Figure 6 is a photograph comparing the analysis of the expression of MSTN protein in MSTN-/-male pigs and natural pigs produced through the secondary somatic cell nuclear transfer and embryo transfer of the present invention by Western blot.
  • Figure 7 is a photograph of the morphological comparison of muscle tissue through the immunohistochemical method in MSTN-/-male pigs and natural pigs produced through secondary somatic cell nuclear transfer and embryo transfer of the present invention.
  • FIG. 8 analyzes 400 muscle fibers by measuring the diameter of muscle fibers using Sc i on Image software in MSTN-/-male pigs and natural pigs produced through secondary somatic cell nuclear transfer and embryo transfer according to the present invention, and their average values This is a chart that calculates and compares.
  • MSTN by mutating pig MSTN gene using TALEN Recombinant expression vectors were constructed to knock out genes.
  • the specific sequence of the MSTN gene to which the TALE domain will bind was selected.
  • the first half-site sequence and the second half-site sequence to which the TALE domain binds are selected. It was. Sequence analysis of pig MSTN gene by bioinformatics method and DNA cleavage ability analysis of TALEN in specific sequence that Tlzen has in database have a length of 42-54 bp in pig MSTN gene and the starting sequence is thymine (T), a specific sequence was selected for the 4 'sequences (SEQ ID NOS: 1 to 4), the last sequence being adenine (A).
  • TALENs were generated for four sequences to measure the activity of TALENs (FIG. 2).
  • the sequence of the first half-site to which the left TALE domain is bound is 5 ⁇ -ttcaaatcctcagtaaaactt-3 ⁇
  • the sequence of the second half-site to which the right TALE domain is bound is 5,-gctcctaacattagcaaaga-.
  • the sequence to be cleaved by the Fokl cleavage domain linked to the TALE domain was cgcctggaaaca.
  • TALENs that will act on specific sequences of selected porcine MSTN genes
  • the method disclosed in the Korean patent application 'Genome engineering through designed TAL effector nuclease' was used.
  • TALE-repeats that specifically bind to each base in the selected particular sequence
  • a TALE domain capable of specifically binding to the entire specific sequence was designed, and the cleavage domain of nucleic acid endonuclease ( Fokl) were allowed to connect.
  • the entire amino acid sequence of TALEN to bind to the first half is shown in SEQ ID NO: 5
  • the entire amino acid sequence of TALEN to bind to the second half is shown in SEQ ID NO: 6.
  • the Pig myostatin TALEN for knockout of the myostatin gene contains 135 ampicillin drug resistance genes, CMV promoter, HA epitope tag, nuclear localization signal, and N-terminal end of AvrBs3. Amino acids, TALE domains that bind to specific sequences of porcine myostatin, and Fokl cleavage domains were arranged in order (FIG. 3). To join in the first half digit. The sequence of the recombinant expression vector for expressing TALEN is shown in SEQ ID NO: 7, the sequence of the recombinant expression vector for expressing TALEN to bind to the second half-digit is shown in SEQ ID NO: 8.
  • SEQ ID NO: 7 The sequence of the recombinant expression vector for expressing TALEN to bind to the second half-digit is shown in SEQ ID NO: 8.
  • Porcine fetal fibroblasts were constructed by subcultured primary fibroblasts obtained from fetuses isolated from adult female pigs 40 days old, of which the cells from the male fetus were taken as donor cells. The conventional method was applied to the process of establishing the cell line. The following related reagents were all used by Gibco Corporation (USA). The sterile membrane was cut under sterile conditions, the pig embryo was placed in PBS, repeatedly marched 3-5 times until the wash solution became clear, then transferred to Petridish, and the head and organs were cut with ophthalmic scissors. Sufficiently washed and sterilized with alcohol, it was again cut into a 0.5 lmm 3 tissue mass.
  • the cut tissue mass was placed in DMEM medium containing 0.25% trypsin and left for 4 minutes in a C02 incubator at 38.5 ° C., followed by addition of DMEM medium containing fetal bovine serum to stop the trypsin activity, and then 15 mfi. Transfer to the test tube was centrifuged for 10 minutes at a rate of 1,500 pi ⁇ . Discard the centrifuged supernatant, add 3 m £ of 20% FBS / DMEM preheated to 38.5 ° C to loosen the mass, spread evenly into a sterile cell culture dish and place in a 38.5 ° C C02 incubator Put incubated. Afterwards, the cells were passaged 3-5 times while observing the morphology and growth of the cells every day, and then used for cell transformation experiments.
  • the pig fetal fibroblasts were 15% fetal bovine blood serum, 1% non-essential amino acid,
  • the ovaries were obtained from female pigs before puberty, and then follicles of 3-8 mm diameter were sucked out of the ovaries with a follicle suction pump. Cultivated, dense, and homogeneous cumulus cell-oocyte complexes (Cu ⁇ Lus-oocyte-complexes, COCs) were selected and washed three times with TALP medium, followed by oocyte in vitro maturation (in vitro maturation media, IVM media). The components of the IVM culture were as follows: 10% pig follicle (pEE) with TCM-199, 0.6 ⁇ l iolLl L-cysteine, 10 IU / m.
  • oocytes selected as receptors were treated for 1 hour in a culture solution containing 0.4 ng / mi demecolcine and 0.05 mol / L sucrose, followed by 5 mg / cytokalacin BCcytochalasin. B) and 0.4 mg / demecolcine were transferred to the solution containing.
  • the micromanipulator (1 ⁇ 71, Olympus, Japan) was used to remove M II oocytes, first pole cells, and progenitor cell nuclei with a nucleus removal needle, and then used for somatic cell nuclear transfer.
  • Example 6 Primary Somatic Cell Nuclear Transplantation by Fusion
  • the oocytes from which the nucleus of Example 5 was removed were equilibrated in a fusion solution (0.28 ⁇ L ol / L mannitol, 0.1 ⁇ L ol / L MgC12), and then placed in an fusion bath to which the fusion solution was applied. .
  • the donor cells of Example 3 were also placed in the fusion tank, and then the donor cell-receptor oocyte membrane contact surface was parallel to the electrode using a glass needle, and then 2 kV / cm, using a fusion device (LF301, BEX, Japan). Fusion was performed by adding a DC current field of 20 mu 5 .
  • the reconstituted egg was incubated for 1 hour in a culture solution containing 0.4 mg / ⁇ demecolcine. Activated by treatment with 1.5 kV / cm, 100 us DC current field in a solution containing activator (0.28 ⁇ ol / L mannitol, 0.1 ⁇ ol / L MgC12) and 0.1 ⁇ ol / L Ca2 +, followed by 2 ⁇ ol / Incubated for 4 hours in a culture medium containing L 6-DMAP.
  • Example 7 Embryo Transfer and Pregnancy Diagnosis of Surrogate Sows
  • the embryo reconstituted by fusion in the above manner was placed in NCSU-37 culture medium,
  • R-18 205 25 3 18-1, 18-2, 18-3
  • Fetal gDNA was extracted from the fetus using the G—DEX II cDNA Extraction Kit (Intronbio, South Korea). The extracted gDNA was used as a template for nested PCR, and the primer was designed to amplify the target site of TALEN for myostatin knockout: forward C-ctggtcccgtggatctgaatg-S '), reverse direction (5'-gatcgtttccgtcgtagcgtg-3) ')
  • the primers were used to obtain a 302 bp PCR product, which was Nested PCR was performed as a template and using the following primers: 7 ⁇ 3 ⁇ 4 " 3 ⁇ 4 (5'—gaatgagaacaacacgcgagcaaagg_3 '), reverse (5' ⁇ catcttccaaggagccatcac-3 ')
  • Second somatic cell nuclear transfer and embryonic transplantation were performed using a cell line established from F2 mutated to two MSTN alleles in the mutant fetus of Example 8 as donor cells. Somatic cell nuclear transfer and embryonic transplantation were carried out in the same manner as the primary somatic cell nuclear transfer and embryo transplantation method of Examples 4 to 7. Transplantation of 8 embryos into 4 surrogate sows yielded 20 piglets (Table 2). The MSTN + cloned pigs produced by this method were pigs of the same age in the control group (2 Month age) showed distinct double muscle characteristics (FIG. 5).
  • biceps brachii were extracted from MSTN— / clone pigs and native pigs, 25 days after birth, respectively.
  • the muscle tissue was fixed in formaldehyde for 10 hours, then dehydrated using an alcohol gradient, then clarified with chloroform, embedded with paraffin, and then sliced with a microtome to a thickness of 3-5 ⁇ a day. Cut.
  • the sliced tissue slices were quickly immersed in a 30% aqueous alcohol solution, and then transferred to a 40 ° C. constant water cooler to completely spread the tissue slices and adhered to the slides.
  • MSTN— ⁇ cloned pigs were found to have an increased diameter of muscle fibers compared to native piglets (FIG. 8).
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Publication number Priority date Publication date Assignee Title
KR20130116306A (ko) * 2011-01-03 2013-10-23 주식회사 툴젠 디자인된 tal 이펙터 뉴클레아제를 통한 게놈 엔지니어링
KR20140046408A (ko) * 2011-02-25 2014-04-18 리컴비네틱스 인코포레이티드 유전자 변형 동물 및 이의 생산방법

Family Cites Families (6)

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CN101892264A (zh) * 2010-05-28 2010-11-24 吉林大学 肌生成抑制素mstn基因敲除猪的建立
CN102021201A (zh) * 2010-09-06 2011-04-20 南京农业大学 一种肌肉生成抑制素基因敲除猪的制备方法
CN102296073B (zh) * 2011-08-11 2014-04-23 中国农业科学院北京畜牧兽医研究所 锌指核酸酶定点敲除Myostatin基因特异靶位点
CN103088044B (zh) * 2011-11-02 2014-09-03 内蒙古大学 一种敲除牛mstn基因的打靶载体及其应用
AR091482A1 (es) 2012-06-21 2015-02-04 Recombinetics Inc Celulas modificadas geneticamente y metodos par su obtencion
PT3494997T (pt) 2012-07-25 2019-12-05 Massachusetts Inst Technology Proteínas de ligação a adn indutíveis e ferramentas de perturbação do genoma e aplicações destas

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130116306A (ko) * 2011-01-03 2013-10-23 주식회사 툴젠 디자인된 tal 이펙터 뉴클레아제를 통한 게놈 엔지니어링
KR20140046408A (ko) * 2011-02-25 2014-04-18 리컴비네틱스 인코포레이티드 유전자 변형 동물 및 이의 생산방법

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
SUNG, YOUNG HOON ET AL.: "Knockout mice created by TALEN-mediated gene targeting", NATURE BIOTECHNOLOGY, vol. 31, no. 1, January 2013 (2013-01-01), pages 23 - 24, XP055597463, DOI: 10.1038/nbt.2477 *
TESSON: "Knockout rats generated by embryo microinjection of TALENs", NATURE BIOTECHNOLOGY, vol. 29, no. 8, 2011, pages 695 - 696, XP002681970, DOI: 10.1038/NBT.1940 *
XU: "Targeted myostatin gene editing in multiple mammalian species directed by a single pair of TALE nucleases", MOLECULAR THERAPY-NUCLEIC ACIDS, vol. 2, no. 7, 2013, pages 1 - 10, XP055184659, DOI: doi:10.1038/mtna.2013.39 *

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