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Zinc finger nuclease mediated MSTN gene mutation sequence and application thereof

A nucleotide sequence and gene technology, applied in the field of porcine MSTN gene mutation sequence

Pending Publication Date: 2015-11-18
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no report of "double muscle" phenotype caused by natural or artificial mutation of MSTN in pigs

Method used

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  • Zinc finger nuclease mediated MSTN gene mutation sequence and application thereof
  • Zinc finger nuclease mediated MSTN gene mutation sequence and application thereof
  • Zinc finger nuclease mediated MSTN gene mutation sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Zinc finger nuclease-directed knockout of MSTN gene

[0057] The wild-type fibroblasts are isolated purebred Meishan pig primary fibroblasts, and the MSTN gene in the genome is the wild-type MSTN gene (shown in sequence 3 in the sequence listing);

[0058] Mutant fibroblasts are the fibroblasts obtained by knocking out the 3781-3791 nucleotides of the MSTN gene in the primary fibroblast genome of the isolated purebred Meishan pig, and the MSTN mutant gene (in the sequence table) shown in sequence 2).

[0059] Mutant fibroblasts were obtained by introducing ZFN plasmids into wild-type fibroblasts.

[0060] The ZFN plasmid is a pair of ZFN plasmids from sigma company, and its specific target site sequence is CCTTCCCAGGACcaggaGAAGATGGGCTGGTA (corresponding to nucleotides 3770-3801 at the 5' end of the MSTN gene shown in sequence 3 in the sequence listing).

[0061] Specific steps are as follows:

[0062] 1. Design and construction of ZFN plasmids

[0063] 1....

Embodiment 2

[0106] Embodiment 2, the application of MSTN mutation gene

[0107] 1. Preparation of mutant fibroblasts by knocking out MSTN gene with TALEN

[0108] The wild-type fibroblasts are isolated purebred Meishan pig primary fibroblasts, and the MSTN gene in the genome is the wild-type MSTN gene (shown in sequence 3 in the sequence listing);

[0109] Mutant fibroblasts are the fibroblasts obtained by knocking out the 3781-3791 nucleotides of the MSTN gene in the primary fibroblast genome of the isolated purebred Meishan pig, and the MSTN mutant gene (in the sequence table) shown in sequence 2).

[0110] Mutant fibroblasts were obtained by introducing TALENmRNA pairs and DonorDNA into wild-type fibroblasts.

[0111] The TALENmRNA pair is a pair of TALENmRNA pair prepared according to the Visunlight Talen kit, and its target site sequence is shown in the 3780-3795th nucleotide molecule of sequence 3 in the sequence listing. The TALEN mRNA pair is obtained by mixing Talen left arm a...

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Abstract

The invention discloses a zinc finger nuclease mediated MSTN gene mutation sequence and application thereof. Base deletion is promoted by mediating porcine MSTN (myostatin) gene by virtue of a zinc finger nuclease technology to cause frameshift mutation, and as a result, MSTN functional protein is not formed since translation is terminated in advance; and meanwhile, a mutation sequence of the MSTN gene that nucleotides on 3781st-3791st sites are deleted is obtained. Tests prove that pigs of nucleotide deletion on 3781st-3791st sites in the MSTN gene have marked double-muscled phenotype as well as significant increase in both muscle content and lean percentage.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to zinc finger nuclease-mediated pig MSTN gene mutation sequence and application thereof. Background technique [0002] Zinc Finger Nuclease (ZincFinger Nuclease, ZFN) is a chimeric protein formed by the recombination of the cleavage domain of zinc finger protein and FokI endonuclease, in which the zinc finger protein can specifically bind to the target sequence of interest, and the FokI endonuclease Enzymes are responsible for the specific cutting of DNA sequences. When the two ZFNs bind to the target target sequence located 5 to 7 bases apart on the double strand of DNA, they can form a dimer, and then activate the function of FokI endonuclease, so that the DNA produces a double at a specific site. Strand breaks, and then the broken double strands are repaired by homologous recombination or non-homologous end joining. Since the variability of non-homologous end-joining repair leads ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12Q1/68C12N15/877A01K67/027
Inventor 崔文涛钱丽丽汤茂学李奎
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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