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Cattle serine/threonine kinase STK11 interfering adenovirus construction method

A threonine kinase, STK11 technology, applied in the field of genetic engineering, can solve the problems of short duration of action of siRNA products, low transfection efficiency of shRNA vector, and difficulty in establishing gene knockout model individuals, so as to achieve good and reliable infection effect. The effect of experimental techniques

Inactive Publication Date: 2019-04-09
NORTHWEST A & F UNIV
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Problems solved by technology

[0004] However, as a large livestock animal, it is difficult to establish a gene knockout model for cattle. If the isolated primary target cells are used as the research object, the general shRNA vector transfection efficiency is low, and the siRNA products synthesized on the market have a short duration of action. Therefore, the study of bovine serine threonine kinase STK11 interference with adenovirus can achieve simultaneous or independent knockdown of the expression of the STK11 gene in bovine adipocytes and muscle cells, laying a good technical foundation for the study of the function of the STK11 gene

Method used

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  • Cattle serine/threonine kinase STK11 interfering adenovirus construction method
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  • Cattle serine/threonine kinase STK11 interfering adenovirus construction method

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Embodiment Construction

[0036] This example provides a method for constructing a bovine serine-threonine kinase STK11-interfering adenovirus. First, the shRNA sequence with the highest interference efficiency is screened, and then the shRNA sequence is recombined with an interfering adenovirus vector to construct an STK11-interfering adenovirus;

[0037] The shRNA sequence is:

[0038] 5'-GACGACGAGCTCTTCGACATTTCAAGAGAATGTCGAAGAGCTCGTCGTCTTTTTTTAGATCTG-3'.

[0039] The sources of vectors, bacterial strains, cells and reagents involved in this embodiment are as follows:

[0040] Bovine preadipocytes were obtained from perirenal fat of Qinchuan cattle from the National Beef Cattle Improvement Center by enzymatic digestion.

[0041] HEK293 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and DH5α was purchased from Tiangen Biochemical Technology Co., Ltd.;

[0042] The RT-PCR kit was purchased from Takara Company, the plasmid extraction and gel recovery kits were pur...

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Abstract

The invention relates to a cattle serine / threonine kinase STK11 interfering adenovirus construction method, an shRNA sequence with the optimal interfering efficiency is firstly screened, then the shRNA sequence is recombined with an interfering adenovirus vector to construct the STK11 interfering adenovirus; the infecting and the interfering efficiency of the STK11 interfering adenovirus are verified in Qinchuan cattle primary preadipocyte, the result shows that the infecting effect is good, the expression of the STK11 on the mRNA level is reduced by 76%, the protein level is also remarkably reduced, continuously knocking down an STK11 gene in a somatic cell is successfully realized, and the reliable experiment technique is provided for conducting the related researches on the function ofthe STK11 gene in future.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for constructing bovine serine threonine kinase STK11 interfering adenovirus. Background technique [0002] Serine / threonine kinase 11 (serine / threonine, kinase, STK11), also known as liver kinase B1 (LKBl), is a key upstream protein molecule that activates the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, involved in cell polarity, cell Various cellular processes such as division, cell proliferation, cell differentiation, and cell metabolism mainly function through two signal transduction mechanisms: phosphorylation and subcellular localization. [0003] Studies have shown that the STK11-AMPK pathway is an important signaling pathway that affects the two major metabolic organs of adipose tissue and muscle tissue. Studies in model animal mice have found that knocking out the adipose tissue STK11 gene affects the development and...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/113
CPCC12N15/1137C12N15/86C12N2710/10143C12N2310/14C12N2800/107C12N2310/531
Inventor 王力昝林森成功张松
Owner NORTHWEST A & F UNIV
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