KR20130069188A - Zinc finger nuclease for targeting myostatin and use thereof - Google Patents

Abstract

PURPOSE: A zinc finger nuclease which targets myostatin is provided to knock out a myostatin gene on a zebra fish genome, and to quickly growth an individual. CONSTITUTION: A zinc finger nuclease which targets myostatin is a fusion protein with a zinc finger domain and a nucleotide cleavage domain and has a cleavage activity by being bound to a myostatin(mstn) gene. The cleaved mstn cannot express C-terminal of myostatin. The zinc finger domain contains 2-10 zinc finger modules containing sequences selected from the group consisting in sequence numbers 1-9. A method for knocking out mstn comprises a step of step the zinc finger nuclease. A method for producing an animal, exclusive of human, with improved growth rate or increased individual size contains mstn-knocked out cells.

Description

Zinc finger nuclease for targeting myostatin and use according to myostatin

The present invention relates to zinc finger nucleases targeting myostatin, and more particularly to the zinc finger nucleases; A polynucleotide encoding the zinc finger nuclease; A vector comprising said polynucleotide; Knocking out myostatin using the zinc finger nuclease; A cell or animal in which the myostatin gene is knocked out by the above method; A kit for knockout of a myostatin gene comprising the zinc finger nuclease, polynucleotide, or vector; And it relates to a method for producing animals other than humans by increasing the growth rate or increase in size than the wild type by the knocking out method.

In 2001, the Human Genome Project was completed and the entire sequence of the human genome was revealed, revealing genes that have not been studied. In order to study the functions of these newly known genes, the necessity of research using animal models as well as cell-level experiments is emerging. In the study of gene function, C. elegans , Drosophila ( D. melanogaster ), Frog ( X. laevis ), Zebrafish ( D. rerio ), Mice ( M. musculus ) and Chimpanzee ( P. troglodytes ) It is mainly used as an animal model. Of these, zebrafish belong to the vertebrate gate, which is systematically close to humans, so that the structure of the gene is similar, and it is easy to control experimentally, and a large number of embryos can be obtained steadily, the development is fast, and the embryo is transparent. Have good benefits in gene function research (Graham JL and Peter DC, Nature) Rev Genet . , May; 8 (5) 353-367, 2007). Zebrafish are the experimental animals mentioned above, and because of their merits, they are animal models selected on demand in gene function studies and developmental studies.

Zinc finger nuclease (Zinc-Finger Nuclease) is a restriction enzyme at the zinc finger domain and the carboxyl terminal (C-terminal, C-terminal) that can recognize the sequence at the amine terminal (N-terminal, N-terminal) It is a chimeric protein combining the cleavage domain of Fok I. Each monomer zinc finger nuclease recognizes and attaches 9-12 specific nucleotide sequences at 5-6 base intervals in the forward and reverse strands. When two zinc finger nucleases are attached to a specific nucleotide sequence, a cleavage domain at the end of the carboxyl group forms a dimer, causing a double-strand break in the nucleotide sequence (Smith J. et al., Nucleic Acids Res . , 27, 674-681, 1999) When cells of multicellular animals undergo such double-stranded damage, they rapidly undergo sequence repair mechanisms such as homologous recombination and non-homologous end joining. DNA repair system). Of these repair mechanisms, non-homologous end-bonds serve as the main repair mechanism, which often results in the loss of or insertion of sequences due to linking the cross-sections without homology or microhomology alone (Kristoffer). V. & Lawrence F P., Oncogene , 22, 5792-5812, 2003). That is, a mutation occurs. In this manner, zinc finger nucleases can be used to knock out genes.

The myostatin (mstn) gene is a regulatory ligand belonging to the transforming growth factor-β superfamily, whose gene structure is similar to other genes belonging to the TGF-β superfamily (Fig. 1A). Myostatin acts as a negative regulator of the development and growth of musculoskeletal structures. In addition to development and growth, it also acts as a negative regulator of muscle homeostasis. Myostatin has been shown to have orthologs in most mammals, birds, fish, etc., and humans and cattle (Ravi Kambadur et al., Genome) Res . , 7: 910-915, 2007), sheep, dogs (Dana S. Mosher et al., Plos genet . , 3: 779-786, 2007), a variation present in nature has been identified, indicating a phenotype in which muscle is overweight than wild-type (FIG. 1B). In mice, when myostatin gene knockouts were heterozygotes, muscles increased in small amounts, and when knockouts were homozygotes, muscle doubled (muscle doubled) increased significantly (Se-Jin Lee et al., Nature , 16; 98). : 9306-9311, 1997). Thus, if the myostatin gene can be knocked out effectively, it will be possible to produce improved fish or livestock that grow rapidly or grow muscles to grow significantly.

Against this background, the inventors have made diligent efforts to find ways to effectively knock out myostatin. As a result, we have targeted the zeofish myostatin 1 (mstn1) gene using zinc finger nuclease technology. It confirmed that it knocked out, and completed this invention.

One object of the present invention is a fusion protein comprising a zinc finger domain and a nucleotide cleavage domain, a zinc finger nuclease targeting a myostatin having an activity of binding to and cleaving a myostatin (mstn) gene. To provide.

Another object of the present invention is to provide a polynucleotide encoding the zinc finger nuclease.

Still another object of the present invention is to provide a vector comprising the polynucleotide.

Still another object of the present invention is to provide a method of knocking out a myostatin gene using the zinc finger nuclease.

Still another object of the present invention is to provide an animal except a human or a cell in which the myostatin gene is knocked out.

Still another object of the present invention is to provide a method of manufacturing an animal except for a human, in which the growth rate or the size of an individual is increased than a wild type, including a cell knocked out of the myostatin gene by the method of knocking out the myostatin gene. It is.

As one aspect for achieving the above object, the present invention is a fusion protein comprising a zinc finger domain and a nucleotide cleavage domain, targets myostatin having the activity of binding to and cleaving myostatin (mstn) gene A zinc finger nuclease is provided.

As used herein, the term “fusion protein” refers to a protein in which two or more different polypeptides are linked into one strand of polypeptide through a peptide bond. The polypeptide has an activity of binding to a desired site in the nucleotide sequence of myostatin and optionally cleaving it, including a zinc finger domain and a nucleotide cleavage domain. In the present specification, a fusion protein may be used interchangeably with zinc finger nuclease or ZFN.

Methods of designing and building such fusion proteins (or polynucleotides encoding fusion proteins) are well known in the art. In one embodiment of the present invention, a polynucleotide encoding a fusion protein comprising a zinc finger domain and a nucleotide cleavage domain was constructed and cloned into a vector plasmid (Example 1).

Generally, the zinc finger domain of the fusion protein is located near the amino terminus (N-terminus) of the fusion protein, and the nucleotide cleavage domain (or cleavage half domain) is located near the carboxy terminus (C-terminus), preferably the fusion of the present invention. The protein also has a zinc finger domain located at the N-terminus and a nucleotide cleavage domain (or cleavage half domain) at the C-terminus.

In the present invention, the term "myostatin (mstn)" may be used interchangeably with "myostatin 1". Specifically, the myostatin may be referred to as myostatin 1 in the case of zebrafish. The myostatin, also known as growth differentiation factor 8 (GDF8), is known as a gene that inhibits muscle differentiation and growth, and more specifically, acts as a negative regulator that regulates the development and growth of musculoskeletal structures and muscle homeostasis in the body. It is a gene. Gene sequences thereof and the like can be obtained from known databases (GenBank et al.). Myostatin is a regulatory ligand belonging to the TGF-β superfamily and its gene structure is similar to other genes belonging to the TGF-β superfamily as shown in FIG. 1A. When the myostatin gene knockout is heterozygous, a small amount of muscle is increased, and when the homozygote is homozygous, the result is that the muscle is doubled and the muscle is greatly increased. Since it is possible to create an increasing number of objects, it is necessary to develop a method of effectively knocking it out, but the effective method is still insufficient. Accordingly, the zinc finger nuclease of the present invention can be cleaved by targeting a specific nucleic acid sequence of myostatin, and when the zinc finger nuclease according to the present invention is used for individual production, by knocking out the intracellular myostatin gene Production can be increased by producing individuals of increased growth and size.

As used herein, the term "targeting myostatin" refers to specifically recognizing and cleaving a specific nucleic acid sequence of myostatin. In the present invention, the zinc finger domain is specific for a specific nucleic acid sequence of myostatin. Recognized as a target, cleavage may be generated in myostatin by a nucleotide cleavage domain linked to the zinc finger domain by a peptide bond. The term “sequence” means a nucleotide sequence regardless of its length, which may be straight, circular or branched DNA or RNA, and may be single helical or double helical.

The zinc finger nuclease (ZFN) is a fusion protein composed of a zinc finger domain that specifically recognizes a DNA sequence of myostatin and a nucleotide cleavage domain that cleaves DNA of myostatin as an artificial restriction enzyme. Means.

The zinc finger domain is a protein that binds to the DNA sequence of the myostatin gene in a manner having sequence specificity through one or several zinc finger modules, and the zinc finger domain may be combined with zinc finger modules. It can be designed to bind to the selected sequence of the ostatin gene.

The zinc finger domain has a structure in which two or more zinc finger modules that recognize DNA 3bp (base pair) are connected. Preferably, the zinc finger module may be linked with an amino acid linker, wherein the amino acid linker may be composed of 3 to 20 amino acids, and preferably 3 to 9 amino acids.

A zinc finger domain that binds to a specific nucleotide sequence of the myostatin gene can be prepared by linking two or more zinc finger modules using an amino acid linker consisting of an appropriate number of amino acids. At this time, by adjusting the number of amino acids of the amino acid linker linking between the zinc finger module and the module appropriately, by adjusting the nucleotide sequence on the myostatin gene, the interval between the nucleotide sequence, etc. that each zinc finger module is bound, Zinc finger domains having the activity of binding to selected sequences of genes can be prepared.

The myostatin gene cut by the zinc finger nuclease may not be able to express the C-terminal protein.

Since each zinc finger module independently recognizes the DNA sequence of myostatin, for example, a zinc finger domain consisting of three or four modules can bind to the 9 or 12bp sequence of myostatin. Thus, for zinc finger nucleases that act as dimers, a pair of zinc finger nucleases, each consisting of three or four zinc finger modules, may specifically recognize 18 to 24 bp.

The zinc finger domain may be prepared by selecting two or more of the zinc finger modules of SEQ ID NOs: 1 to 9. That is, two or more zinc finger modules are selected from 33 zinc finger modules (SEQ ID NOS: 1 to 9) having specific DNA binding specificities to prepare zinc finger domains, and a fusion comprising the produced zinc finger domain and nucleotide cleavage domain. By producing a protein, a zinc finger nuclease having an activity of binding to and cleaving a myostatin gene can be produced.

The zinc finger domain may include two or more zinc finger modules selected from the group consisting of SEQ ID NOs: 1 to 9, preferably 2 to 10 zinc finger modules, and more Preferably, it may include two to four zinc finger modules, more preferably three or four zinc finger modules.

In addition, the zinc finger domain may be any one zinc finger domain selected from the group of six zinc finger domains composed of the zinc finger modules described in Table 1.

The zinc finger module refers to an amino acid sequence inside a domain whose structure is stable during coordination with zinc ions, and may be nine zinc finger modules represented by amino acid sequences of SEQ ID NOs: 1 to 9 derived from humans and the like. (FIG. 6).

In order to fabricate the zinc finger nuclease targeting the myostatin, a proper type and number of zinc finger modules are selected in consideration of the target sites of the nine zinc finger modules described in FIG. 6 to identify the myostatin gene. Zinc finger domains having the activity of binding to the nucleotide sequence can be prepared.

Preferably, the zinc finger domain may comprise a zinc finger module represented by SEQ ID NOs: 3, 5, and 9, and more preferably, a zinc finger represented by SEQ ID NOs: 3, 5, and 9 from the N-terminus of the fusion protein. Module, and even more preferably, a zinc finger domain represented by the amino acid sequence of SEQ ID NO.

In addition, preferably, the zinc finger domain may include a zinc finger module represented by SEQ ID NOs: 1, 2, 4, and 6, and more preferably, SEQ ID NOs: 1, 2, 4, and 6 from the N-terminus of the fusion protein. It may include a zinc finger module represented by, and even more preferably may be a zinc finger domain represented by the amino acid sequence of SEQ ID NO: 15.

Further, preferably, the zinc finger domain includes a domain including a zinc finger module represented by SEQ ID NOs: 3, 5, and 7, a domain including a zinc finger module represented by SEQ ID NOs: 3, 5, 8, and 9, SEQ ID NO: 3 , And a zinc finger nuclease selected from the group consisting of a domain comprising a zinc finger module represented by 5, 7, and 8, and a domain comprising a zinc finger module represented by SEQ ID NOs: 2, 4, and 6.

In one embodiment of the present invention, among the six zinc finger domains (Table 1) prepared, a zinc finger nu target that targets myostatin 1 including a zinc finger module represented by SEQ ID NOs: 3, 5 and 9 ZFN e1_219a pair, which is a zinc finger nuclease (e1_219F) that targets myostatin 1, including a clease (e1_219Ra) and a zinc finger module represented by SEQ ID NOs: 1, 2, 4, and 6, and SEQ ID NO: 3, Myostatin comprising a zinc finger nuclease (e1_219Rb) targeting myostatin 1 comprising a zinc finger module represented by 5 and 7 and a zinc finger module represented by SEQ ID NOs: 1, 2, 4 and 6 ZFN e1_219b pair, a zinc finger nuclease (e1_219F) targeting 1, was transfected into reporter cells and analyzed for SSA (single strand annealing) activity, resulting in at least 40% activity (FIG. 3B). Appropriate to It was confirmed that the ZFN. In addition, after ZFN e1_219a pair was microinjected into zebrafish, genomic DNA was isolated to perform T7E1 mismatch detection analysis. As a result, small fragments of DNA amplified from cells treated with the ZFN e1_219a pair were cleaved by T7E1, confirming that the ZFN e1_219a pair targets myostatin 1 (FIG. 4).

In addition, analysis of the nucleotide sequence of the DNA amplified in the zebrafish injected with the ZFN e1_219a pair was confirmed that mutation was induced on the myostatin 1 gene by the ZFN e1_219a pair targeting the myostatin 1 ( 5). These results indicate that the zinc finger nuclease that targets myostatin 1 of the present invention can knock out the myostatin 1 gene with high efficiency. Suggest.

As used herein, the term “cleavage” means to unlink the covalently bonded backbone of the myostatin nucleotide molecule, and the “nucleotide cleavage domain” refers to a polypeptide sequence having enzymatic activity for nucleotide cleavage of such myostatin. Means.

The nucleotide cleavage domain can be obtained from an endonuclease or exonuclease. Examples of endonucleases from which nucleotide cleavage domains can be obtained include, but are not limited to, restriction endonucleases, recurrent endonucleases, and the like. 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. In this sense, a cleavage domain that cleaves all double-bonded nucleotide sequences is also used as a cleavage half domain.

Such restriction endonucleases are present in many classes of species and are capable of sequence specific binding with DNA (recognition sites) and can cleave DNA in the vicinity of binding sites. Certain restriction endonucleases, such as type IIs, have a binding domain and a cleavage domain that are separated by cleaving DNA even at sites where the recognition site has been removed. For example, the type IIs enzyme FokI 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.

The nucleotide cleavage domain may be derived from a type IIs restriction endonuclease, and the type IIs restriction endonuclease is not limited thereto, but FokI , AarI, AceIII , AciI , AloI , BaeI , Bbr7I , CdiI , CjePI , EciI , Esp3I , FinI , MboI , sapI or SspD51 , preferably FokI .

In addition, the zinc finger nucleases of the present invention may further comprise a nuclear localization signal sequence. The nuclear position signal can be used as a means for moving the zinc finger nuclease into the nucleus for knocking out the myostatin gene by the zinc finger nuclease targeting the myostatin of the present invention. Preferably, the nuclear position signal may be a nuclear position signal represented by the amino acid sequence of SEQ ID NO: 18 (PPKKKRKV).

In addition, the zinc finger nuclease of the present invention may be a zinc finger nuclease represented by the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17.

In another aspect, the present invention provides a polynucleotide encoding a zinc finger nuclease according to the present invention.

The polynucleotide is a polymer of nucleotides in which nucleotide monomers are long chained by covalent bonds, and are strands of DNA or ribonucleic acid (RNA) or RNA (ribonucleic acid) having a predetermined length or more. Polynucleotides encoding clease.

The polynucleotide may be delivered intracellularly in a known form, for example, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, which has been used as a conventional gene carrier. Viral vectors, including liposomes, polylysine, polyethylenimine (PEI), protamine, histones, polyesteramines and their respective variants In addition to cationic polymers, non-viral vectors such as micelles, emulsions, nanoparticles, and the like can be used, and can be efficiently delivered into cells by utilizing known intracellular material transfer peptides in addition to vector systems.

As another aspect, the invention provides a vector comprising a polynucleotide encoding a zinc finger nuclease according to the invention.

The vector may be introduced into a cell to express the zinc finger nuclease of the present invention, and a known expression vector such as a plasmid vector, a cosmid vector, a bacteriophage vector, and the like may be used. According to known methods of the art can be easily prepared.

The vector of the present invention may be a recombinant vector operably linked to a polynucleotide encoding a zinc finger nuclease according to the present invention. "Operably linked" refers to that expression control sequences are linked to regulate transcription and translation of the polynucleotide sequence encoding the zinc finger nuclease, wherein the polynucleotide sequence is expressed under the control of the expression control sequence (including the promoter) Maintaining an accurate reading frame such that zinc finger nucleases encoded by the polynucleotide sequence are produced.

As another aspect, the present invention provides a method for knocking out the myostatin (mstn) gene using the zinc finger nuclease according to the present invention.

In still another aspect, the present invention provides an animal except a human or a cell in which the myostatin gene is knocked out by a method of knocking out the myostatin gene according to the present invention.

The cell may be a cell or an animal in which one or two alleles of the myostatin gene is knocked out, preferably a cell derived from an animal other than a human being, or an animal, to increase the yield of the individual.

In the present invention, the term "animal except human" is an individual who wants to increase the production of the individual, but is not limited thereto, and may be a mammal, a fish, a bird, and the like. Preferably may be fish. In one embodiment of the present invention used zebrafish.

In the present invention, the term "knockout" means inhibiting the expression of the myostatin gene, and in the present invention, mutations in the myostatin gene are knocked out by a zinc finger nuclease that targets myostatin. do.

Zinc finger nucleases can function in the form of dimers, either in the form of a homodimer or a heterodimer, and the double strands can be cleaved to achieve the desired object of the present invention. Zinc finger nucleases that function as dimers consist of two ZFN monomers in order to recognize a single DNA site of interest. In addition, since one zinc finger module recognizes and binds DNA 3bp, a zinc finger domain consisting of two to four zinc finger modules may bind to a DNA recognition region of 6 to 12 bp to form a ZFN pair. .

Preferably, the method for knocking out the myostatin gene of the present invention comprises the steps of (a) expressing the zinc finger nuclease according to the present invention intracellularly or extracellularly and introducing into the cell (b) the The zinc finger nuclease expressed or introduced in the cell may cause double strand damage to the myostatin gene in the cell, and (c) the mutation may be induced in the myostatin gene by the double stranded damage. .

Incorporation of the zinc finger nuclease into the cell may preferably introduce one or more pairs of zinc finger nucleases into the cell in order to cause double strand damage to the myostatin gene, and more preferably, SEQ ID NO: A pair of zinc finger nucleases comprising a zinc finger domain represented by an amino acid sequence and a zinc finger nuclease comprising a zinc finger domain represented by an amino acid sequence of SEQ ID NO: 15 may be introduced into a cell, and even more preferred. Preferably, a pair of zinc finger nucleases represented by the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 17 can be introduced into the cell.

The zinc finger nuclease may be introduced in the form of a polypeptide or polynucleotide, and the method for this may be any method known in the art. For example, foreign DNA may be introduced into a cell by transfection or transduction in order to introduce the zinc finger nuclease into the cell. Transfection includes calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroshock, microinjection, liposome fusion, lipofectamine and protoplast fusion. It can be carried out by various methods known in the art.

In addition, the expression in the cells of the zinc finger nuclease can be any method known in the art, for example, a vector can be used. Such vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors may include signal sequences for secretion in addition to expression control factors such as promoters, operators, initiation codons, termination codons, polyadenylation sequences and enhancers, and can be prepared in various ways according to the purpose.

The cell is a cell to knock out the myostatin gene by the zinc finger nuclease of the present invention, prokaryotic cells such as E. coli; Eukaryotic cells such as yeast, fungi, protozoa, higher plants, insects; Amphibian cells; Fish cells; Mammalian cells such as CHO, HeLa, COS-1, HEK, HCT116, K562, BJ fibroblast, such as cultured cells (in vitro), grafts and primary cultures (in vitro and ex vivo) and cells in vivo; Or cells derived from blood and various tissues isolated from human donors and patients, but are not limited thereto. For the purposes of the present invention may be an individual to increase the production.

When the zinc finger nuclease is introduced into a cell, the zinc finger nuclease can induce a double stranded damage to a desired portion on the myostatin gene. To repair the damaged area. At this time, the cell repairs the damaged site by non-homologous end joining (NHEJ) method, and an error-prone that causes insertion or deletion at the broken DNA strand ends occurs. Repair is performed in the direction of). Thus, mutations in the myostatin gene can be induced, ultimately knocking out the myostatin gene.

Preferably, the mutation may be a mutation by substitution, deletion, insertion and combination thereof.

In one embodiment of the present invention, as a result of using ZFN e1_219a pair typically among zinc finger nucleases of the present invention to knock out myostatin 1 gene, myostatin in zebrafish microinjected with ZFN e1_219a pair Mutation was induced in the 1 gene, it was confirmed that the myostatin 1 gene ultimately knocked out (Fig. 5). These results suggest that the zinc finger nuclease of the present invention can be usefully used to make cells in which myostatin 1 gene is knocked out, and that the growth of fast and large individuals can be made.

In another aspect, the invention provides a myostatin comprising a zinc finger nuclease according to the invention, a polynucleotide encoding said zinc finger nuclease, or a vector comprising said polynucleotide. mstn) provides a knockout kit for genes.

In addition, the kit for knockout of the myostatin gene may further include an instruction manual describing an optimal reaction performance condition. Instructions for use include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the brochure may include information that is disclosed or provided through an electronic medium, such as the Internet.

The kit for knockout of the myostatin gene is a zinc finger nuclease that targets an appropriate amount of myostatin required for knockout of the myostatin gene, a polynucleotide encoding the zinc finger nuclease, or the polynucleotide It may include a vector comprising a.

In another embodiment, the present invention provides an animal except for a human having an improved growth rate or increased individual size than a wild type, including a cell knocked out of a myostatin gene using a zinc finger nuclease according to the present invention. Provide a way to. The animal may preferably be a fish.

The preparation of such animals can be carried out by introducing a zinc finger nuclease according to the present invention into a fertilized egg of a embryonic state by a known method. Or by introducing a zinc finger nuclease according to the present invention into somatic cells by a known somatic cell nuclear transfer method.

As used herein, the term "nuclear transfer" refers to transplanting the nucleus of a cell into an egg from which the nucleus has already been removed, and an individual born by implanting such a nucleated fertilized egg has a genetic material of a donor cell. It is a genetically identical clone because it has been delivered to the nuclear donor cytoplasm.

Methods for removing genetic material of eggs include physical methods, chemical methods, centrifugation using Cytochalasin B, etc. (Tatham et al., Hum Reprod., 11 (7); 1499-1503, 1996). Somatic cells knocked out of the myostatin gene are introduced into the nucleus from which the nucleus has been removed by cell membrane fusion, intracellular microinjection, or the like. 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 recombined by fusion by changing the viscosity of the cell membrane through electrical stimulation. At this time, it is convenient to use an electric fusion machine that can freely adjust the fine current and voltage.

The zinc finger nuclease targeting the myostatin of the present invention can be used to knock out the myostatin gene on the zebrafish genome, thereby making it possible to prepare an individual whose growth is fast or large. In addition, knockout of intracellular myostatin by the zinc finger nuclease of the present invention can be maintained as long as the cell is alive, and is delivered as it is to daughter cells during cell division. Can be used effectively.

1 is a diagram showing the structure of the myostatin gene and animals lacking myostatin. (A) shows the structure of the myostatin gene, and (B) shows the animals lacking the myostatin gene. a. dog; b. belgian blue; c. mice; d.
Figure 2 is a diagram showing the amino acid sequence of the ZFN pair and e1_219a ZFN pair that can target myostatin 1 gene (SEQ ID NO: 16 and 17). A ZFN pair capable of targeting zebrafish myostatin 1 gene was designed with ZF module of Tulcene. It is a figure which shows the amino acid sequence of e1_219a ZFN pair. Bold: amino acid sequence that recognizes a specific base of the ZF module.
3 is a diagram showing the mechanism of luciferase-SSA reporter assay and the results of the activity test of myostatin ZFN. (A) is a complete lucifer by SSA DNA repair mechanism when double stranded damage occurs due to a ZFN attached to a vector cloning the base sequence of myostatin ZFN between incomplete fragments designed to overlap the intermediate 300bp of luciferase. It is a figure showing that the activity can be measured by being expressed as a laze. (B) is a diagram showing the relative activity value for I-SceI when ZFN targeting the zebrafish myostatin gene is treated. Zif268 represents zinc finger protein present in nature.
Figure 4 is a view showing the principle of the surveyor nuclease mismatch assay and confirmation of mutations in the subject microinjected with e1_219a ZFN. In order to confirm the target mutation of e1_219a ZFN, the target DNA of e1_219a ZFN was amplified, and the product was heterozygous formed by re-unfolding to confirm the mutation by cutting with T7endonuclease1. In the gel plot, we can see the cut band of the expected size in the sample microinjected with the e1_219a ZFN pair.
Figure 5 is a diagram showing the identification of the nucleotide sequence and protein amino acid sequence changes by e1_219a ZFN. (A) is a diagram showing the nucleotide sequence of the mutation confirmed by sequencing analysis in the sample microinjected with e1_219a ZFN. (B) is a figure which shows the change of the amino acid sequence by nucleotide sequence change (red arrowhead: target position of e1_219a ZFN).
6 shows a zinc finger module and its target sequence.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  One: Zinc Finger  Nuclease ( ZFN Design and vector production

<1-1> Zinc Finger  Nuclease ( ZFN ) design

In order to prepare ZFNs, ZF modules that can be attached to 9-12bp each have a 5-6bp spacing on the forward and reverse strands of DNA.

Thus, ZFN combination list for each exon DNA sequence of zebrafish myostatin 1 (mstn1) gene was selected based on the ZF module possessed by ToolGen, Inc., and is shown in Table 1 below. Indicated.

ZFN name Locus Zinc finger module e1_219a
e1_219Ra RDHT (16) HSSR (28) ISNR (5) e1_219F rdnq (18) DSAR2 (3) RDER2 (15) DSNRa (1) e1_219b
e1_219Rb RDHT (16) VSSR (22) ISNR (5) e1_219F rdnq (18) DSAR2 (3) RDER2 (15) DSNRa (1) e1_222a
e1_222Ra RDHT (16) HSSR (28) ISNR (5) VSTR (23) e1_222F rdnq (18) DSAR2 (3) RDER2 (15) e1_222b
e1_222Rb RDHT (16) VSSR (22) ISNR (5) VSTR (23) e1_222F rdnq (18) DSAR2 (3) RDER2 (15)

Using the ToolGen ZFNfinder program, a computer algorithm program developed by Tulgen for the development of multi-finger zinc pink nucleases, the portions to which each ZFN module is attached, the recognized sequences, and the combined modules Output.

<1-2> ZFN  Production of expression vector

Each 1-finger module of ZFNs is inserted into the expression vector pcDNA3.1 (Invitrogen) vector. Vector of ZF (Zinc-Finger) which is located in the N- terminal restriction enzyme PstI and AgeI (5'-A ^ CCGGT- 3 ') vector of ZF for cutting, and the C- terminal position in the PstI and XmaI (5 '-C ^ CCGGG-3') was isolated by gel extraction kits (QIAquick Gel Extraction Kit, QIAGEN). The isolated DNA fragment was ligated with T4 ligase to construct a two-finger nuclease vector. Again, a 3- / 4-finger nuclease vector was constructed in the same manner as above (Bae, KH et al., Nat Biotechnol. , 21, 275-280, 2003). Each ZFN paste was cloned after a single ZF domains and consists of the cutting of the module 3-4 and one restriction enzyme FokI, the restriction enzyme AgeI and ZF module using the complementarity of the cut surface of the XmaI recognizing 3bp continuously.

<1-3> ZFN of Fok Mutant  substitute

By cutting the 3/4-finger nucleases vector of ZFN with AgeI and XhoI to remove the template DNA fragment Gel extraction kits (QIAquick Gel Extraction Kit , QIAGEN) and, FokI sharkey variant also cleaved with XhoI and AgeI template DNA The pieces were separated by Gel extraction kits (QIAquick Gel Extraction Kit, QIAGEN). The DNA fragment was attached with T4 ligase. The linked fragments were cloned into pcDNA3 (Invitrogen).

Example  2: SSA  ( Single strand annealing ) Reporter system ZFN  Activity analysis

As a method of determining ZFN activity, a bacterial two-hybridization (B2H) system (JK Joung, et al., Proc . Natl . Acad . Sci . , 97, 7382-7387, 2000; JA Hurt, et al., Proc . Natl . Acad. Sci., 100, 12271-12276, 2003) and SSA (Single-strand annealing) system (Chames P, et al., Nucleic Acids Res . , 33: e178. doi: 10.1093 / nar / gni 175, 2005; Y. Doyon, et al., Nature biotech . , 26, 702-708, 2008). We independently designed and constructed an SSA reporter system in mammalian cells to evaluate the activity of ZFN. SSA is one of the pathways for repairing double-strand breakage (DSB) and complementarily repairs DNAs when the same nucleotide sequence is repeated in close proximity to the site where the DSB occurred (FIG. 3A). That is, when double stranded damage occurs due to a ZFN attached to a vector cloning the target nucleotide sequence of myostatin 1 ZFN between incomplete fragments in which intermediate 300bp of luciferase is designed to overlap, complete luciferase is induced by SSA DNA repair mechanism. Since it is expressed, it is a principle that can measure the activity. The cell-based luciferase-SSA system was constructed in the following manner and ZFN activity was measured.

<2-1> Luciferase SSA  Vector production

The pcDNA5 / FRT / TO- luciferase- SSA vector was digested with BamHI , and the mstn1 gene inserted into the pGEM T-easy vector was digested with BamHI , isolated by Gel extraction kits (QIAquick Gel Extraction Kit, QIAGEN), and then T4 ligase. Ligation to prepare pcDNA5 / FRT / TO- luciferase -SSA-mstn1.

<2-2> Transduction stable cell line  making

Flp-In T-REx 293 cells (KDR biotech co., LTD) were treated with 10% FBS (fetal bovine serum), 100 units / ml penicillin, 100 μg / ml streptomycin and 0.1 mM nonessential amino acids (NEAA). Culture was put in DMEM (Dulbecco's modified Eagle's medium) containing. The Flp-In T-REx 293 Cell 8 × 10 ⁵ cells / ㎖ to spread (ratio 1 of pcDNA5 / FRT / TO- luciferase -SSA- mstn1 and pOG44 (Flp recombinase expression vector): 9) of the 3 DNA ㎍ the Lipoic After transfection with Pectamine 2000 (Invitrogen), the cells were incubated at 37 ° C. 5% CO ₂ for ₂ days. The cultured cells were treated with hygromycin at a final concentration of 200 μg / ml, and the vectors inserted into the genome of the cells were selected to produce stable cell lines, which were used as reporter cells.

<2-3> SSA  System-based ZFN  Activity analysis

After culturing the reporter cells in which pcDNA5 / FRT / TO- luciferase- SSA-mstn1 was inserted in Example <2-2> in a 96-well plate, a plasmid expressing ZFN of each ZFN pair was prepared. 100 ng each was transfected using Lipofectamine 2000 (Invitrogen). When cultured 48 hours after transfection, 1 μg / ml of doxycycline was treated to induce luciferase gene expression. After 24 hours of incubation, the cells were recovered and lysed in 20 μl of 1 × lysis buffer (Promega). Luciferase activity was measured by adding 2 μl of cell lysate to 10 μl of luciferase assay reagent (Promega).

As a result, when the activity of the control (control) I-SceI was set to 100%, the activity of the e1_219a and e1_219b pairs was found to be about 40% or more (FIG. 3B). Results using ZFN pairs with an activity of about 15-57% (Hye-Joo Kim, et al., Genome research , 19 (7): 1279-1288, 2009), confirmed the appropriate ZFN for inducing mutations.

Example  3: ZFN Mutation detection by

<3-1> Zebra fish  Occurrence and Generation  Ready

The reporter system analysis of Example 2 was to analyze the mutation of the most active ZFN pair e1_219a ZFN (right: FokI sharkeyRR, left finger: FokI sharkeyDAS).

The zebrafish, a small tropical fish, was bred in a fishbowl maintained at a water temperature of 28.5 ° C, and the food was used by hatching commercial brine shrimp. Since zebrafish scattering and fertilization are induced by light, they maintain a contrast of 14 hours during the day and 10 hours at night. In the afternoon of the previous day, each male and female is put together in a dedicated mating cage, and after 10 hours of cancer treatment, fertilized eggs are born the next day. The fertilized eggs were collected and washed with Ringer's solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl ₂ , 5 mM HEPES, pH7.2), transferred to Petri dishes and generated in an incubator at 28.5 ° C. The developmental process was observed by anatomical microscope over time.

<3-2> in vitro transcription ( In vitro transcription )

To synthesize the sense-mRNA of each ZFN with good activity through the SSA system, it was digested with restriction enzyme PvuII to cut the 3 'back of the poly (A) signal and the portion where the ZFN module was encoded, and with ethanol (EtOH). Purification was made. Sense-mRNA was synthesized using mMESSAGE mMACHINE ™ (Ambion) in the linearized plasmid.

<3-3> mRNA  Micro injection

After obtaining the first cell-phase fertilized egg of a zebrafish-producing embryo, the ZFN pair mRNA prepared in Example <3-2> was applied to the cytoplasm or yolk of the fourth-cell fertilized egg at the first cell phase. Microinjection using World Precision Instruments). The final concentration of mRNA was mixed 1: 1 with 0.5% phenol red so that the final concentration was 200 ng / μl and about 100 pg ~ 1 ng was injected into one fertilized egg.

<3-4> genome DNA  detach

In order to isolate genomic DNA (genome DNA), the cells were cultured for 2 ~ 3 days after microinjection, followed by DNA extraction buffer (10 mM Tris, pH8.0, 200 mM NaCl, 10 mM EDTA, 0.5% SDS, 100 ㎍ /). ㎖ Proteinase K) was added and reacted at 55 ° C for 12 hours. The embryonic lysate was extracted with phenol / chloroform, purified with ethanol, dissolved in 1 × TE (10 mM Tris, pH8.0, 1 mM EDTA), and genomic DNA was isolated.

<3-5> ZFN  Target position amplification and Rewinding  ( reannealing )

To amplify the mstn1 exon1 ZFN target moiety, a first PCR was performed using genomic DNA as a template with primary oligo primer sets (SEQ ID NOs: 10 and 11) to amplify the ZFN target moiety on the genome. Diluted amplified PCR product by a fifth was amplified by performing a secondary PCR using a secondary oligo primer set (SEQ ID NO: 12 and 13) as a template. The product amplified by the secondary PCR was subjected to the below rewinding setup with a PCR machine. Primers and PCR conditions used for this amplification and re-unfolding are as follows:

Primary Oligo-Fp: 5'-ATAGAGTGGCCAAAGTTGC-3 '(SEQ ID NO: 10)

Primary Oligo-Rp: 5'-TTATGGTCGACGCTTGTGC-3 '(SEQ ID NO: 11)

Secondary Oligo-Fp: 5'-CCTTTAGCACGCCTTGGAA-3 '(SEQ ID NO: 12)

Secondary oligo-Rp: 5'-CTGCGTAAAGGGTCTCTCCA-3 '(SEQ ID NO: 13)

The first PCR conditions were performed at 95 ° C. for 5 minutes, followed by 25 cycles of 95 ° C. 30 seconds, 55 ° C. 30 seconds, and 72 ° C. 30 seconds, followed by 10 minutes at 72 ° C.

Secondary PCR conditions were performed at 95 ° C. for 5 minutes, followed by 35 cycles of 95 ° C. 30 seconds, 60 ° C. 30 seconds, and 72 ° C. 30 seconds, followed by 10 minutes at 72 ° C.

The re-unwinding conditions were performed at 95 ° C. for 2 minutes, and then the temperature was lowered from 95 ° C. to 85 ° C. at a rate of −2 ° C./sec and then to 85 ° C. to 25 ° C. at a rate of −0.1 ° C./sec.

<3-6> T7E1 ( T7endonuclease1 ) mismach  analysis

Surveyor nucleases recognize and cut nicks that do not complement the binding of heteroduplex DNA. Thus, genomic DNA isolated from the individual microinjected with e1_219a ZFN in order to perform T7E1 mismatch analysis was amplified by PCR by the method described in Example 3, and the DNA was unfolded to prepare a heterodimer. The heterodimeric DNA was treated with T7E1 enzyme (Tulgen) for 15 minutes and then loaded onto an agarose gel for analysis.

As a result, no band was cut in the wild type (WT) sample without microinjection of e1_219a ZFN, and the band was cut at about 240 bp of the expected size in the sample microinjected with e1_219a ZFN (Fig. 4). These results support the ZFN of the present invention to specifically recognize and cleave mstn1.

Example  4: In zebrafish ZFN Mutation sequence identification induced by

In order to confirm the ZFN mutations, ZFN target portions of the specimens identified by the surveyor nuclease mismatch assay were amplified by PCR, and the products amplified by PCR using the T-blunt cloning kit (Solgent, Inc.) were analyzed. Inserted as -blunt vector. T7 primers in the plasmid into which the base sequence of the target portion of ZFN was inserted were requested by a sequencing company (Solgent, Inc.) to obtain nucleotide sequence information with mutations.

As a result of the sequencing analysis, it was confirmed that the mutation occurred at the target position of the e1_219a ZFNs (FIG. 5). The identified mutations were confirmed that the 4bp inserted mutations and 4bp deleted mutations that occur mainly in ZFN. These mutations cause the myostatin gene to fail to express normal protein molecules and make short proteins at the N-terminus of the protein. When myostatin proteins become active in cellular signal transduction systems, the C-terminal amino acid polymer (polypeptide) is separated from the N-terminus and acts as a ligand. The mutation identified in the present invention loses myostatin gene function since no C-terminal that serves as a ligand of myostatin is made at all.

These results suggest that the ZFN of the present invention can specifically knock out myostatin 1, suggesting that the individuals thus prepared can produce fast or largely grown individuals.

<110> Toolgen Incorporation <120> Zinc finger nuclease for targeting myostatin and use <130> PA110864 / KR <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> DSNRa <400> 1 Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Asp Ser Ser Asn Leu   1 5 10 15 Gln Arg His Val Arg Asn Ile His              20 <210> 2 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> DSAR2 <400> 2 Tyr Ser Cys Gly Ile Cys Gly Lys Ser Phe Ser Asp Ser Ser Ala Lys   1 5 10 15 Arg Arg His Cys Ile Leu His              20 <210> 3 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> ISNR <400> 3 Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Ile Ser Ser Asn Leu   1 5 10 15 Gln Arg His Val Arg Asn Ile His              20 <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> RDER2 <400> 4 Tyr His Cys Asp Trp Asp Gly Cys Gly Trp Lys Phe Ala Arg Ser Asp   1 5 10 15 Glu Leu Thr Arg His Tyr Arg Lys His              20 25 <210> 5 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> RDHT <400> 5 Phe Gln Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu   1 5 10 15 Lys Thr His Thr Arg Thr His              20 <210> 6 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> rdnq <400> 6 Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe Met Arg Ser Asp Asn Leu   1 5 10 15 Thr Gln His Ile Lys Thr His              20 <210> 7 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> VSSR <400> 7 Tyr Thr Cys Lys Gln Cys Gly Lys Ala Phe Ser Val Ser Ser Ser Leu   1 5 10 15 Arg Arg His Glu Thr Thr His              20 <210> 8 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> VSTR <400> 8 Tyr Glu Cys Asn Tyr Cys Gly Lys Thr Phe Ser Val Ser Ser Thr Leu   1 5 10 15 Ile Arg His Gln Arg Ile His              20 <210> 9 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> HSSR <400> 9 Phe Lys Cys Pro Val Cys Gly Lys Ala Phe Arg His Ser Ser Ser Leu   1 5 10 15 Val Arg His Gln Arg Thr His              20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1st-oligo-Fp primer <400> 10 atagagtggc caaagttgc 19 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1st-oligo-Rp primer <400> 11 ttatggtcga cgcttgtgc 19 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 2nd-oligo-Fp primer <400> 12 cctttagcac gccttggaa 19 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 2nd-oligo-Rp primer <400> 13 ctgcgtaaag ggtctctcca 20 <210> 14 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> e1_219Ra <400> 14 Met Val Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Glu Leu Pro Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Gly Ile Arg Ile Pro Gly Glu Lys Pro Phe Gln              20 25 30 Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr          35 40 45 His Thr Arg Thr His Thr Gly Glu Lys Pro Phe Lys Cys Pro Val Cys      50 55 60 Gly Lys Ala Phe Arg His Ser Ser Ser Leu Val Arg His Gln Arg Thr  65 70 75 80 His Thr Gly Glu Lys Pro Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe                  85 90 95 Ser Ile Ser Ser Asn Leu Gln Arg His Val Arg Asn Ile His Thr Gly             100 105 110 Glu Lys         <210> 15 <211> 144 <212> PRT <213> Artificial Sequence <220> <223> e1_219F <400> 15 Met Val Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Glu Leu Pro Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Gly Ile Arg Ile Pro Gly Glu Lys Pro Phe Ala              20 25 30 Cys Pro Glu Cys Pro Lys Arg Phe Met Arg Ser Asp Asn Leu Thr Gln          35 40 45 His Ile Lys Thr His Thr Gly Glu Lys Pro Tyr Ser Cys Gly Ile Cys      50 55 60 Gly Lys Ser Phe Ser Asp Ser Ser Ala Lys Arg Arg His Cys Ile Leu  65 70 75 80 His Thr Gly Glu Lys Pro Tyr His Cys Asp Trp Asp Gly Cys Gly Trp                  85 90 95 Lys Phe Ala Arg Ser Asp Glu Leu Thr Arg His Tyr Arg Lys His Thr             100 105 110 Gly Glu Lys Pro Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Asp         115 120 125 Ser Ser Asn Leu Gln Arg His Val Arg Asn Ile His Thr Gly Glu Lys     130 135 140 <210> 16 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> e1_219Ra ZFN <400> 16 Met Val Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Glu Leu Pro Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Gly Ile Arg Ile Pro Gly Glu Lys Pro Phe Gln              20 25 30 Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr          35 40 45 His Thr Arg Thr His Thr Gly Glu Lys Pro Phe Lys Cys Pro Val Cys      50 55 60 Gly Lys Ala Phe Arg His Ser Ser Ser Leu Val Arg His Gln Arg Thr  65 70 75 80 His Thr Gly Glu Lys Pro Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe                  85 90 95 Ser Ile Ser Ser Asn Leu Gln Arg His Val Arg Asn Ile His Thr Gly             100 105 110 Glu Lys Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu         115 120 125 Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile Glu     130 135 140 Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val Met 145 150 155 160 Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly Gly                 165 170 175 Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp             180 185 190 Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu         195 200 205 Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn Gln     210 215 220 Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro 225 230 235 240 Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe Lys                 245 250 255 Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys             260 265 270 Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met         275 280 285 Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn     290 295 300 Asn Gly Glu Ile Asn Phe Leu Asp 305 310 <210> 17 <211> 342 <212> PRT <213> Artificial Sequence <220> <223> e1_219F ZFN <400> 17 Met Val Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Glu Leu Pro Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Gly Ile Arg Ile Pro Gly Glu Lys Pro Phe Ala              20 25 30 Cys Pro Glu Cys Pro Lys Arg Phe Met Arg Ser Asp Asn Leu Thr Gln          35 40 45 His Ile Lys Thr His Thr Gly Glu Lys Pro Tyr Ser Cys Gly Ile Cys      50 55 60 Gly Lys Ser Phe Ser Asp Ser Ser Ala Lys Arg Arg His Cys Ile Leu  65 70 75 80 His Thr Gly Glu Lys Pro Tyr His Cys Asp Trp Asp Gly Cys Gly Trp                  85 90 95 Lys Phe Ala Arg Ser Asp Glu Leu Thr Arg His Tyr Arg Lys His Thr             100 105 110 Gly Glu Lys Pro Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Asp         115 120 125 Ser Ser Asn Leu Gln Arg His Val Arg Asn Ile His Thr Gly Glu Lys     130 135 140 Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu Arg His 145 150 155 160 Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile Ala                 165 170 175 Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val Met Glu Phe             180 185 190 Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly Gly Ser Arg         195 200 205 Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly     210 215 220 Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile 225 230 235 240 Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg                 245 250 255 Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser             260 265 270 Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn         275 280 285 Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn Gly     290 295 300 Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys 305 310 315 320 Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly                 325 330 335 Glu Ile Asn Phe Leu Asp             340 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Nuclear localization signal <400> 18 Pro Pro Lys Lys Lys Arg Lys Val   1 5

Claims (23)

A fusion protein comprising a zinc finger domain and a nucleotide cleavage domain, a zinc finger nuclease that targets a myostatin having an activity of binding to and cleaving a myostatin (mstn) gene.
The zinc finger nuclease of claim 1, wherein the truncated myostatin gene does not express the C-terminus of myostatin.
The zinc finger nuclease of claim 1, wherein the zinc finger domain comprises two to ten zinc finger modules selected from the group consisting of SEQ ID NOs: 1-9.
The zinc finger nuclease of claim 1, further comprising a nuclear localization signal sequence.
The zinc finger nuclease of claim 1, wherein the zinc finger domain is selected from the group of zinc finger domains composed of the zinc finger modules of Table 1. 8.
The zinc finger nuclease according to claim 1, wherein the zinc finger domain comprises a zinc finger module represented by SEQ ID NOs: 3, 5, and 9.
The zinc finger nuclease according to claim 6, wherein the zinc finger nuclease is represented by the amino acid sequence of SEQ ID NO.
The zinc finger nuclease of claim 1, wherein the zinc finger domain comprises a zinc finger module represented by SEQ ID NOs: 1, 2, 4, and 6. 6.
The zinc finger nuclease according to claim 8, wherein the zinc finger nuclease is represented by the amino acid sequence of SEQ ID NO.
According to claim 1, wherein the zinc finger domain is a domain comprising a zinc finger module represented by SEQ ID NO: 3, 5 and 7, a domain comprising a zinc finger module represented by SEQ ID NO: 3, 5, 8 and 9, sequence A zinc finger nuclease selected from the group consisting of a domain comprising a zinc finger module represented by numbers 3, 5, 7 and 8, and a domain comprising a zinc finger module represented by SEQ ID NOs: 2, 4 and 6.
The zinc finger nuclease of claim 1, wherein the nucleotide cleavage domain is derived from a Type IIs restriction endonuclease.
The zinc finger nuclease of claim 11, wherein the type IIs restriction endonuclease is FokI .
A polynucleotide encoding the zinc finger nuclease of any one of claims 1 to 12.
A vector comprising the polynucleotide of claim 13.
A method of knocking out a myostatin gene using the zinc finger nuclease of claim 1.
The method of claim 15, wherein the method is
(a) expressing or intracellularly expressing the zinc finger nuclease of any one of claims 1 to 12 into a cell;
(b) the zinc finger nuclease expressed or introduced in the cell causes double strand damage to the myostatin gene in the cell; And
(c) causing the mutation in myostatin by the double stranded damage.
The method of claim 16, wherein the mutation is a mutation by any one or more selected from the group consisting of substitutions, deletions, and insertions.
A cell whose myostatin gene has been knocked out by the method of claim 15.
Animals other than humans whose myostatin gene is knocked out by the method of claim 15.
20. The animal of claim 19, wherein said animal is a fish.
A kit for knockout of the myostatin gene comprising the zinc finger nuclease of claim 1, a polynucleotide of claim 12, or a vector of claim 14.
A method for producing an animal other than a human, including a cell knocked out of the myostatin gene by the method of claim 15, wherein the growth rate is increased or the size of the individual is increased than that of the wild type.
The method of claim 22, wherein the animal is fish.

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