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Porcine myostatin gene editing site 864-883 and its application

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

Active Publication Date: 2019-09-10
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 864-883 and its application
  • Porcine myostatin gene editing site 864-883 and its application
  • Porcine myostatin gene editing site 864-883 and its application

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. 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.

[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 sequen...

Embodiment 2

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

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

[0053] 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.

[0054] (2) Operation steps

[0055] - Cell plating:

[0056] 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.

[0057] - Digestive cells:

[0058] A...

Embodiment 3

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

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

[0073] 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 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 in particular relates to a porcine myostatin gene editing site and 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 cells, beca...

Claims

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Application Information

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Patent Type & Authority Patents(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