Preparation method for myostatin knock-out pig

A technology of myogenesis inhibition and gene knockout, applied in the field of genetic engineering, can solve the problems of less research on genetic engineering, and achieve the effects of difficult technical safety, simple operation, and high practical value in production

Inactive Publication Date: 2011-04-20
NANJING AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

Knocking out the pig Myostatin gene through gene targeting technology has not been reported yet, and using RNAi technology to silence the gene is a...

Method used

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  • Preparation method for myostatin knock-out pig
  • Preparation method for myostatin knock-out pig
  • Preparation method for myostatin knock-out pig

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Embodiment Construction

[0034] (1) Construction of pBluescript-U6 vector containing human U6 promoter

[0035]The U6 promoter sequence was amplified from the human blood genome (TaKaRa) by PCR technology, and the primers and PCR procedures used were referred to the literature (Ohkawa, J. and K. Taira, Control of the functional activity of an antisense RNA by a tetracycline-responsive derivative of the human U6 snRNA promoter. Hum Gene Ther, 2000; 11(4): 577-85.), the obtained PCR product and pBluescriptⅡ KS(+) plasmid (purchased from Stratagene) were carried out with EcoRI and ClaI (TaKaRa) Double enzyme digestion, enzyme digestion products were separated on agarose gel and the required fragments were recovered, and then the recovered insert fragment (U6 promoter) and pBluescriptⅡ KS (+) backbone fragment were mixed in a molar ratio of 3:1, and the T4 Under the action of ligase (TaKaRa), connect overnight at 16°C, then take 10 microliters of the ligated product and transform it into JM109 competent c...

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Abstract

The invention discloses a preparation method for a myostatin knock-out pig, belonging to the technical field of biology. The preparation method is as follows: generating virus particles capable of expressing shRNA of myostatin gene of a targeted pig in a 293Ft cell; then infecting the fibroblasts of a piglet by using the produced slow virus; wherein after 48 hours, the expression of Myostatin genes in the fibroblasts is inhibited by fluorescence quantitative polymerase chain reaction (PCR) detection. In the invention, the slow virus particles can be used for carrying out microinjection on perivitelline space of a fertilized egg of the pig, can lead the virus nucleic acid to be integrated to an embryo gene group so as to express the shRNA, and can produce a transgenic pig with the myostatin of being knocked out after embryo transplantation. The slow virus particles also can be used for producing the transgenic pig by using a sperm vector method. By utilizing the preparation method, the cumbersome operation of knocking out the homologous targeting gene in the traditional method can be avoided, the virus consumption is less and the stable passage can be achieved.

Description

technical field [0001] The invention relates to a method for preparing myostatin gene knockout pigs by using lentiviral vector to mediate RNAi, and belongs to the technical field of genetic engineering. Background technique [0002] my country is the world's largest pig-raising country and the largest pork consumer. But what is not commensurate with it is that the modern pig breeding work is relatively backward, so far there is no modern commercial lean meat pig breed that is fully domesticated (Zhen Yunxiao. Pork import and export situation and countermeasures [J]. China Animal Husbandry Communication, 2003, (12); Wang Aiguo. Review and prospect of my country's pig industry [J]. Contemporary Animal Husbandry, 2004, (01).). Therefore, cultivating high lean meat rate and grain-saving pig breeds is a major demand of the country, and it is also a key and difficult point in the field of pig genetics and breeding. The development of transgenic and gene knockout technology provid...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/113C12N15/87A61D19/02A01K67/027
Inventor 刘红林邹晓龙姜保春
Owner NANJING AGRICULTURAL UNIVERSITY
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