Method for screening effective shRNA of lipoprotein lipase gene

A technology of lipoprotein lipase and screening method, which is applied in the field of screening effective shRNA of lipoprotein lipase gene, to achieve intuitive and reliable results, time-saving and high-efficiency screening process, time-saving and cost-saving effects

Inactive Publication Date: 2010-10-13
NORTHWEST A & F UNIV
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But up to now, it is still blank to screen the effec...

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  • Method for screening effective shRNA of lipoprotein lipase gene
  • Method for screening effective shRNA of lipoprotein lipase gene
  • Method for screening effective shRNA of lipoprotein lipase gene

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Embodiment Construction

[0026] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0027] Step 1: Design and synthesis of single-stranded DNA oligonucleotide fragments encoding the target shRNA;

[0028] According to the LPL sequence of Xinong Saanen sheep published by GenBank, three LPL-siRNAs and one negative control sequence were designed using the online siRNA selection program (http: / / jura.wi.mit.edu / bioc / siRNAext / home.php) , namely siRNA-435: GGCGGATGAATTTAACTAT (SEQ ID NO: 1); siRNA-876: CCGGTGCAATTCCAAAGAA (SEQ ID NO: 2); siRNA-1150: CCTGAAGTCTCCACAAATA (SEQ ID NO: 3); negative control siRNA-NC: ACTACCGTTGTTATAGGTG (SEQ ID NO: 4). On the basis of siRNA, the 19bp oligonucleotides were combined in forward and reverse directions, and the hairpin loop sequence of GAGTACTG (containing Sca I restriction site for identification in vector construction) was added in the middle to make the inside of the oligonucleotide A ha...

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Abstract

The invention discloses a method for screening an effective shRNA of a lipoprotein lipase gene, which comprises the following steps of: A1, designing and synthesizing the singe-chain DNA oligonucleotides fragment for coding target shRNA; A2, constructing a carrier of pENTR/CMV-GFP/U6-shRNA; A3: constructing a carrier of pDsRed1-C1-LPL; and A4, screening effective sequences of the shRNA. The interference carrier and target gene are marked by using red fluorescent light and green fluorescent light respectively, so that the screening process of interference sequences is time-saving and efficient, the result is more intuitionistic and more reliable, and a plurality of sequences can be screened simultaneously.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for screening effective shRNA of lipoprotein lipase gene. Background technique [0002] RNA interference is a genetic interference phenomenon mediated by double-stranded RNA (dsRNA), which can specifically and effectively degrade mRNA, thereby causing gene silencing. Since it was first reported in the journal Nature in 2001 that the specific expression silencing of target genes was successfully induced by siRNA in mammalian cells, RNA interference technology has been widely used as a specific gene silencing tool in mammalian cells. In 2003, Rubinson et al. reported that RNA interference was successfully achieved in primary cultured cells, stem cells and transgenic mice with a virus system, which greatly expanded the scope of application of this technology. At present, RNAi has been developed into a genetic tool for regulating biological gene expression, widely u...

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 赵旺生罗军王伟滕炎玲孙雨婷
Owner NORTHWEST A & F UNIV
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