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A zinc finger nuclease-mediated pig mstn gene mutation sequence and its application

A sequence, gene technology, applied in the biological field

Active Publication Date: 2018-05-15
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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  • A zinc finger nuclease-mediated pig mstn gene mutation sequence and its application
  • A zinc finger nuclease-mediated pig mstn gene mutation sequence and its application
  • A zinc finger nuclease-mediated pig mstn gene mutation sequence and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. Zinc finger nuclease-directed knockout of MSTN gene

[0064] The wild-type fibroblasts are primary fibroblasts from purebred Meishan pigs in vitro, and the MSTN gene in the genome is the wild-type MSTN gene (shown in sequence 2 in the sequence listing);

[0065] Mutant fibroblasts are fibroblasts obtained by knocking out nucleotides 3782-3796 of the MSTN gene in the genome of primary fibroblasts from purebred Meishan pigs and mutating the 3800th nucleotide molecule from T to G A cell, the cell contains the MSTN mutant gene (shown in sequence 1 in the sequence listing).

[0066] Mutant fibroblasts were obtained by introducing ZFN plasmids into wild-type fibroblasts.

[0067] 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 2 in the sequence listing).

[0068] Specific steps are as foll...

Embodiment 2

[0113] Embodiment 2, the application of MSTN mutation gene

[0114] 1. Preparation of mutant fibroblasts by knocking out MSTN gene with TALEN

[0115] The wild-type fibroblasts are primary fibroblasts from purebred Meishan pigs in vitro, and the MSTN gene in the genome is the wild-type MSTN gene (shown in sequence 2 in the sequence listing);

[0116] Mutant fibroblasts are fibroblasts obtained by knocking out nucleotides 3782-3796 of the MSTN gene in the genome of primary fibroblasts from purebred Meishan pigs and mutating the 3800th nucleotide molecule from T to G A cell, the cell contains the MSTN mutant gene (shown in sequence 1 in the sequence listing).

[0117] Mutant fibroblasts are obtained by introducing TALEN mRNA pairs and Donor DNA into wild-type fibroblasts.

[0118] The TALEN mRNA pair is a pair of TALEN mRNA prepared according to the Visunlight Talen kit, and its target site sequence is shown in the 3780-3795th nucleotide molecule of Sequence 2 in the sequence ...

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Abstract

The invention discloses a zinc finger nuclease-mediated porcine MSTN (myostatin) gene mutation sequence and application thereof. By mediating the porcine MSTN gene by the zinc finger nuclease technique, base deletion is enabled, frameshift mutation is resulted in, early termination of translation is caused, an MSTN functional protein is unable to be formed, and the MSTN gene mutation sequence with 3782th to 3796th nucleotides deleted and 3800th nucleotide molecules mutated from T to G is acquired. Experiments show that a pig with the MSTN gene having the 3782th to 3796th nucleotides deleted and the 3800th nucleotide molecules mutated from T to G has evident double-muscled phenotype, both muscle content and lean percentage are evidently increased, and fat content is evidently lowered.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a pig MSTN gene mutation sequence mediated by zinc finger nuclease and its application. Background technique [0002] Zinc Finger 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 the target sequence of interest, FokI nucleic acid Endonucleases 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 to the unpred...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6888C12Q1/6858A01K67/027
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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