Cell clone with MyoG (myogenin) gene knock-in and MSTN (myostatin) gene knock-out prepared by Crispr/Cas9 technique

A gene knockout and gene knock-in technology, applied in the field of genetic engineering, can solve problems such as affecting the number of muscle fibers and affecting the quality of meat

Inactive Publication Date: 2019-09-20
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

All of the above studies have shown that the MyoG gene is a gene related to the number of muscle fibers, and

Method used

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  • Cell clone with MyoG (myogenin) gene knock-in and MSTN (myostatin) gene knock-out prepared by Crispr/Cas9 technique
  • Cell clone with MyoG (myogenin) gene knock-in and MSTN (myostatin) gene knock-out prepared by Crispr/Cas9 technique
  • Cell clone with MyoG (myogenin) gene knock-in and MSTN (myostatin) gene knock-out prepared by Crispr/Cas9 technique

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Experimental program
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Embodiment 1

[0020] 1. Experimental plan

[0021] 1) Construction of MSTN targeting vector

[0022] (1) Design and annealing of target site primers: According to the predicted target site site ( http: / / zifit.partners.org / ZiFiT / CSquare 9Nuclease.aspx) to predict the target site of the mature protein coding region of MSTN gene, and design corresponding primers for the second exon and third exon of MSTN according to the prediction results (see Table 1 for specific primers). The corresponding primers were diluted to a final concentration of 50nM, reacted at 94°C for 3min, incubated at 37°C for 1h, and annealed.

[0023] Table 1 The corresponding sgRNA primers designed according to the predicted target

[0024]

[0025] (2) Single enzyme digestion of psPgRNA and pX330 plasmids and recovery of carrier fragments: extract psPgRNA and pX330 using a plasmid extraction kit, absorb 1 ng each after measuring the concentration with a UV spectrophotometer, and perform single enzyme digestion wi...

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Abstract

The invention belongs to the technical field of genetic engineering and relates to a cell clone with MyoG (myogenin) gene knock-in and MSTN (myostatin) gene knock-out prepared by Crispr/Cas9 technique. The cell clone is characterized in that after being isolated and purified, the cell clone may be induced by 2% horse serum to differentiate into muscle fibers; skeleton satellite cells herein can merge into myotubes; an MSTN gene knock-out vector target is connected successfully, with no base mutations occurring; experimental group cells can differentiate into a certain quantity of myotubes; it is indicated that after MyoG gene is interfered, the cells still can differentiate into myotubes, while total myotube fusion rate is reduced and the form and quantity are significantly differed from those of a control group.

Description

technical field [0001] The invention relates to preparing MyoG gene knock-in and MSTN gene knock-out cell clones by using Crispr / Cas9 technology, and belongs to the technical field of genetic engineering. Background technique [0002] Transgenic technology has become an important means of modern animal breeding technology. It is recognized as the fourth generation of genetic research after linkage analysis in the early 20th century, somatic cell inheritance in the 1960s and gene cloning technology in the 1970s. A turning point in the history of science development. Traditional animal breed improvement can only be carried out between species of the same species or closely related species, and natural mutations are used as the prerequisite for selection, and the probability of such natural mutations is quite low. Transgenic technology can overcome the above problems, create new mutations or break the limitations of gene exchange between species, and speed up the process of an...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/85C12N15/12
CPCC07K14/4702C07K14/4703C12N15/85C12N15/907C12N2800/107C12N2810/10
Inventor 李树峰佟慧丽严云勤
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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