Directional knockout on sheep MSTN gene and detection method for influence thereof on myogenic differentiation

A technology of myogenic differentiation and detection method, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, analytical materials, etc., can solve the problems of low efficiency, cumbersome experiments, high toxicity, etc., and achieve small toxic effects and simple methods Ease of operation and less non-specific shearing effect

Inactive Publication Date: 2016-08-03
YANGZHOU UNIV
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Problems solved by technology

[0003] Aiming at the shortcomings of traditional ENN technology, such as cumbersome experiments, low efficiency, and high t

Method used

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  • Directional knockout on sheep MSTN gene and detection method for influence thereof on myogenic differentiation
  • Directional knockout on sheep MSTN gene and detection method for influence thereof on myogenic differentiation
  • Directional knockout on sheep MSTN gene and detection method for influence thereof on myogenic differentiation

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0032] 1. Construction of CRISPR / Cas9 gene knockout vector

[0033] (1) Target gene cloning: Query the NCBI database to obtain the CDS sequence of the sheep target gene (ID: NM_001009428), use Primer5.0 software to design specific primers, and use the cDNA formed by reverse transcription of the total RNA of Hu sheep muscle tissue as a template for PCR Clone and sequence to obtain the entire CDS sequence of the target gene, purify and recover it and send it to the company for sequencing, and obtain the complete exon sequence and mutation site information of the target gene through comparison;

[0034] (2) gRNA design and synthesis: avoid mutation sites, search for PAM sequences in the exon region, and design target sequences about 20 bp before the PAM sequence, all target sequences are compared in the whole genome, and there is no target homology The point is used as gRNA and synthesized;

[0035] (3) Construction of CRISPR / Cas9 gene knockout vector: the synthetic gRNA was con...

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Abstract

The invention discloses directional knockout on sheep MSTN gene by applying the CRISPR/Cas9 technology, and a method for verifying the impact effect of directional knockout on skeletal muscle satellite cell differentiation. The concrete contents are as follows: firstly, target gene cloning; secondly, gRBA design and synthesis; thirdly, CRISPR/Cas9 gene knockout vector construction; fourthly, CRISPR/Cas9 gene knockout vector exogenous activity detection; fifthly, CRISPR/Cas9 gene knockout vector endogenous activity detection; sixthly, CRISPR/Cas9 gene knockout vector knockout effect detection. The method has the following advantages: firstly, the experimental period is shorter; secondly, the method is simple and practicable, has high repeatability, and can be performed in common laboratories; thirdly, active vector constructed through the method is small in toxic and side effects, and can be applied to the preparation and production of transgenic animals; fourthly, the method is small in non-specific shear, and obviously improves the gene targeting knockout efficiency; fifthly, the method is high in applicability.

Description

Technical field: [0001] The invention relates to a detection method for directional knockout of sheep MSTN gene and its influence on myogenic differentiation, and belongs to the fields of molecular biology, genetic engineering, transgenic technology and the like. technical background: [0002] At present, the research on the function of negative feedback regulatory genes is mainly to use artificial endonuclease (Engineered endonuclease, EEN) technology to knock out genes and carry out functional verification. ) and TALEN (Transcription activator-like effector nuclease, TALEN), however, ZFN technology has poor cleavage specificity and high cytotoxicity due to the homodimeric form with low specificity. As the second-generation EEN technology, TALEN has improved in terms of specific cleavage, but it still has high cytotoxicity. The experimental process of these two technologies is cumbersome and the design is also relatively complicated. The new CRISPR / Cas9 system, as the thir...

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Application Information

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IPC IPC(8): C12Q1/66C12Q1/44G01N33/68
CPCC12Q1/66C12Q1/44G01N33/68G01N2333/47
Inventor 李碧春汤贝贝张亚妮王颖洁左其生李东纪艳芹连超王飞路镇宇
Owner YANGZHOU UNIV
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