Constructing method of goat Myostatin gene knockout carrier

A gene knockout and construction method technology, applied in the field of gene knockout vectors, can solve the problem of low homologous recombination in somatic cells

Inactive Publication Date: 2012-07-18
ANHUI AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

Gene targeting in somatic cells has been successful, but the probability of homologous recombination in somatic cells is low, usually only 10 -6 ~10 -7

Method used

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  • Constructing method of goat Myostatin gene knockout carrier

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Embodiment Construction

[0011] 1. Materials

[0012] Anhui white goat fetal fibroblasts were provided by the laboratory of our school; the targeting vector pLOXP was provided by the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences; pMD18-T vector, high-fidelity long-fragment Tap polymerase and DNA Marker standard, restriction endonuclease, That is, XhoI, SaLI, NotI and BamHI and T4 DNA ligase were purchased from Dalian Bao Biological Engineering Co., Ltd.; plasmid extraction kit was purchased from QIAGEN; Escherichia coli DH5a, cell genomic DNA extraction kit, gel purification recovery kit purchased from Beijing Tiangen Technology Biochemical Co., Ltd. A large amount of endotoxin-free plasmid DNA extraction kit was purchased from QIAGEN; transfection reagent Basic kit was purchased from LONZA company.

[0013] 2. Method

[0014] (1) Primer design and synthesis

[0015] Since there is no sequence report of goat MSTN gene, the primers for PCR ...

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Abstract

The invention discloses a constructing method of a goat Myostatin gene knockout carrier. In the method, according to sheep Myostatin gene sequence, two pairs of PCR (Polymerase Chain Reaction) primers are designed, genome DNA (Deoxyribonucleic Acid) extracted from goat fetus fibroblast is used as a template, amplification is carried out by adopting high-fidelity long-fragment Taq enzyme and a Long-PCR method to obtain homologous arms, wherein the homologous long and short arms are 4.6kb and 1.9kb, respectively. In order to verify whether the sequence of the obtained homologous arms can be used or not, the amplified fragments are respectively cloned to a pMD18-T carrier, and sequencing and homology comparison are carried out after enzyme digestion and PCR identification; and then the homologous long and short arms are respectively cloned to a carrier pLOXP and then identified by enzyme digestion and RCR method, and screening is carried out to obtain a MNST gene targeting carrier pLOXP-MSTN containing neo and HSV-tk positive-negative screening marker genes. According to the invention, the Myostatin gene knockout carrier is constructed by the Long-PCR method, and goat fetus fibroblast is transfected to obtain gene knockout cells; and by adopting the technique, animals with high lean meat percentage can be obtained, and a MNST gene knockout goat model is constructed, so that the method has important significance in further researching the function of the gene in goat.

Description

[technical field] [0001] The invention relates to a gene knockout vector, in particular to a method for constructing a goat Myostatin gene knockout vector. [Background technique] [0002] Myostatin, or MSTN, also known as "growth differentiation factor 8", is a member of the TGF-β superprotein family and has the function of inhibiting skeletal muscle differentiation. MSTN is related to the regulation of the total amount of skeletal muscle in animals, and the loss of its function will cause abnormal hypertrophy of skeletal muscle. In cattle, a mutation in the conserved functional region of MSTN gene is closely related to meat production. Both homozygous and heterozygous beef cattle with this mutation show the advantages of muscular development, increased birth weight and fast growth speed. The weight of skeletal muscle in MSTN knockout mice prepared by gene targeting is more than twice that of normal mice, but the proportion of fat does not increase accordingly, indicating t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/09C12N5/073
Inventor 任春环张子军程箫黄桠锋郭晓飞陈家宏骆先虎刘洪瑜刘旭光
Owner ANHUI AGRICULTURAL UNIVERSITY
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