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Porcine myostatin gene editing site and application thereof

A myostatin and editing technology, applied in the fields of biotechnology and biomedical engineering, can solve the problems of high cost, limited popularization and application, and complicated ZFN preparation process

Active Publication Date: 2016-11-09
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex preparation process and high cost of ZFN, and its technical patents are controlled by a few commercial companies, the promotion and application are very limited.

Method used

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  • Porcine myostatin gene editing site and application thereof
  • Porcine myostatin gene editing site and application thereof
  • Porcine myostatin gene editing site and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of Cas9gRNA expression vector T11 inserted with porcine myostatin gene (MSTN) editing target site T1

[0040] (1) Sources of main reagents and materials

[0041] The pX330-U6-Chimeric_BB-CBh-hSpCas9 (Mammalian Expression, CRISPRhumanized S.pyogenes Cas9) vector was purchased from Addgene, and the targeting vector dT1 was designed by our laboratory. Both AgeI and EcoRI restriction enzymes were purchased from Fermentas. The recombinant vector was carried out in accordance with the instructions of the small-scale ultra-pure plasmid extraction kit provided by Beijing Tiangen Biotechnology Co., Ltd. DNA tapping gel recovery kit was purchased from Qiagen. Sequencing was entrusted to Shenzhen Huada Gene Co., Ltd. to complete.

[0042] (1) The Cas9gRNA expression vector uses the purchased pX330-U6-Chimeric_BB-CBh-hSpCas9 vector as the backbone, and then inserts the required guide sequence according to the fixed oligo design pattern. The guide sequence...

Embodiment 2

[0055] Screening of pig transgenic cell lines after electroporation transfection of embodiment 2

[0056] (1) Sources of main reagents and materials

[0057] The porcine kidney PK15 cell line was purchased from ATCC. The preparation of endotoxin-free plasmids was carried out according to the instructions of the small-scale ultra-pure plasmid extraction kit provided by Beijing Tiangen Biotechnology Co., Ltd. G418 antibiotic was purchased from Sigma. DMEM, DPBS, fetal bovine serum, DMSO were purchased from Invitrogen. The electrotransfer buffer was prepared by the laboratory itself. BTX In 2001, the cell electric fusion instrument was purchased from BTX Company of the United States.

[0058] (2) Operation steps

[0059] - Cell plating:

[0060] The day before electroporation, PK15 cells were digested with trypsin to form a single-cell suspension, followed by 0.25-1×10 6 Cells / well were plated in 6-well plates and grown overnight.

[0061] - Digestive cells:

[0062] A...

Embodiment 3

[0076] Example 3 Confirmation of Selectable Marker Insertion Position and Statistical Analysis of Targeting Efficiency

[0077] (1) Sources of main reagents and materials

[0078] TaKaRa LA Taq and its matching buffer (10×buffer), dNTP, MgSO 4 All were purchased from Toyobo Biotechnology Co., Ltd. Mammalian Genomic DNA Extraction Kit was purchased from Kangwei Century. Sequencing was entrusted to Shenzhen Huada Gene Co., Ltd. to complete.

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Abstract

The invention discloses a porcine myostatin gene editing site and application thereof, and belongs to the fields of biotechnology and biomedical medicine engineering. A gene editing target site is separated from a porcine myostatin gene editing area, the sequence of the gene editing target site is 5'-GGCTGTGTAATGCATGTATGTGG-3', and the gene editing target site is located at a first exon of an MSTN (myostatin) coding area. The site can be recognized by Cas9 endonuclease specifically, so that double strands are medicated to break, homologous recombination between the double strands and shooting carriers is achieved, and mutant genes or selectable maker genes are integrated to a pre-positioned site of a recipient cell genome. A statistical result shows that shooting efficiency of the site is 80.5%. The porcine myostatin gene editing site has the advantages that an effective drone is provided to accurate porcine myostatin gene editing, a new strategy is provided to development of new species of live pigs with high lean meat percentage, and reliable measures and materials are provided to study on molecular mechanism and signal channel of myostatin.

Description

technical field [0001] The invention belongs to the fields of biotechnology and biomedical engineering, and specifically relates to a porcine myostatin gene editing site and an application thereof. Background technique [0002] Gene transfer technology can be divided into two categories according to the site specificity of exogenous gene integration, one is non-site-specific, that is, the site where the introduced exogenous gene is integrated in the target cell genome is generally random, including microinjection , electroporation, calcium phosphate precipitation, retroviral vector infection, etc.: the other type is site-specific, that is, the exogenous gene is introduced into the target cell and integrated into a predetermined site or the intracellular target site is modified site-specifically. It is a gene targeting technology that relies on homologous recombination. It has always been extremely difficult to accurately knock exogenous genes into the genome of pig somatic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/85
CPCC07K14/495C12N15/85C12N2800/107C12N2800/80
Inventor 毕延震刘西梅华文君华再东任红艳李莉肖红卫张立苹郭冬郑新民
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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