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Pig muscle myostatin gene editing site 864-883 and application thereof

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

Active Publication Date: 2017-05-31
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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  • Pig muscle myostatin gene editing site 864-883 and application thereof
  • Pig muscle myostatin gene editing site 864-883 and application thereof
  • Pig muscle myostatin gene editing site 864-883 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of Cas9 gRNA expression vector T21 inserted with porcine myostatin gene (MSTN) editing target site T2

[0037] (1) Sources of main reagents and materials

[0038] The pX330-U6-Chimeric_BB-CBh-hSpCas9 (Mammalian Expression, CRISPRhumanized S.pyogenes Cas9) vector was purchased from Addgene, and the targeting vector dT2 was designed by our laboratory. AgeI and EcoRI restriction endonucleases were purchased from Fermentas. The recombinant vector was carried out according to the instructions of the small-scale medium-volume ultrapure plasmid extraction kit provided by Beijing Tiangen Biotechnology Co., Ltd. The DNA tapping recovery kit was purchased from Qiagen Company. The sequencing was commissioned by Shenzhen Huada Gene Co., Ltd.

[0039] (1) The Cas9 gRNA 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 seq...

Embodiment 2

[0051] Example 2 Screening of pig transgenic cell lines after electroporation transfection

[0052] (1) Sources of main reagents and materials

[0053] Porcine kidney PK15 cell line was purchased from ATCC. The preparation of endotoxin-free plasmids was carried out in accordance with the instructions of the small and medium-volume ultrapure plasmid extraction kit provided by Beijing Tiangen Biotechnology Co., Ltd. The G418 antibiotic was purchased from Sigma. DMEM, DPBS, fetal bovine serum, and DMSO were purchased from Invitrogen. The electro-transfer buffer is prepared by the laboratory. BTX The 2001 cell electrofusion instrument was purchased from BTX Company, USA.

[0054] (2) Operation steps

[0055] -Cell plating:

[0056] The day before electroporation, after trypsinizing PK15 cells into a single cell suspension, follow the steps of 0.25-1×10 6 Cells / wells are spread on 6-well plates and grown overnight.

[0057] -Digest cells:

[0058] Take appropriate trypsin digestion accor...

Embodiment 3

[0071] Example 3 Confirmation of Selective Marker Insertion Position and Statistical Analysis of Targeting Efficiency

[0072] (1) Sources of main reagents and materials

[0073] TaKaRa LA Taq and its supporting buffer (10×buffer), dNTP, MgSO 4 All were purchased from Toyobo Biotechnology Co., Ltd. The mammalian genomic DNA extraction kit was purchased from Kangwei Century. The sequencing was commissioned by Shenzhen Huada Gene Co., Ltd.

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Abstract

The invention discloses a pig muscle myostatin gene editing site 864-883 in a pig genome and application thereof, and belongs to the field of biological engineering. One gene editing target site is separated out from a pig muscle myostatin gene region; the sequence of the target site is 5'-GGATTTTGAAGCTTTTGGATGGG-3'; the site is positioned at a No.3 exon of an MSTN (myostatin) editing region. The site can be specifically identified by Cas9 endonuclease, so that mediated double chains fracture and then generate homologous recombination with targeting vectors; mutant genes or selectable maker gene and the like are integrated to the preset sites of a recipient cell genome. Statistical results show that the site targeting efficiency is 86.7 percent. The pig muscle myostatin gene editing site provided by the invention has the advantages that the effective target is provided for the precise embedding of the gene; new strategies are provided for developing live pig new varieties with high meat factor; reliable means and materials can be provided for developing myostatin molecular mechanisms and signal channels.

Description

Technical field [0001] The invention belongs to the field of biotechnology and bioengineering, and specifically relates to a porcine myostatin gene editing site and its application. Background technique [0002] Gene transfer technology can be divided into two categories according to the site specificity of foreign gene integration. One is non-targeted, that is, the integration site of the introduced foreign gene in the target cell genome is generally random, including microinjection , Electroporation, calcium phosphate precipitation, retroviral vector infection, etc.: The other is site-specific, that is, the foreign gene is introduced into the target cell and integrated into a predetermined site or site-specific modification of the target site in the cell. It is the gene targeting technology that relies on homologous recombination. It has always been extremely difficult to accurately knock foreign genes into the genome of pig somatic cells, because the early gene targeting tech...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/85C12N15/65
Inventor 毕延震刘西梅郑新民李奎任红艳唐中林张立苹陈建武华文君华再东李莉肖红卫
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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