Method for inhibiting target gene expression by antisense lncRNA-mediated cis-regulation

A target gene and antisense technology, applied in the field of molecular biology, can solve the problems of non-sustainable suppression, silencing, and non-persistence

Inactive Publication Date: 2018-09-18
JILIN UNIV
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Problems solved by technology

However, in vitro synthesis of siRNA is expensive, and effective siRNA needs to be identified. The transient expression inhibition of siRNA in cells is not persistent and cannot be used for long-term gene suppression. Due to the limitation of transfection efficiency and difficult cell lines, the use of siRNA has certain limitations. limitations, so many researchers only use RNAi as a tool to study gene function
Pol III promoters can now be used to express short hai

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  • Method for inhibiting target gene expression by antisense lncRNA-mediated cis-regulation
  • Method for inhibiting target gene expression by antisense lncRNA-mediated cis-regulation
  • Method for inhibiting target gene expression by antisense lncRNA-mediated cis-regulation

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Experimental program
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Embodiment Construction

[0015] 1. Selection of target sites

[0016] According to the target gene sequence design, select the target site that the PAM prespacer sequence is adjacent to the motif CRISPR Cas9-gRNA complex specifically recognizes and binds in the downstream sequence of the target gene promoter, including a PAM composed of three nucleotides NGG and Complementary binding to gRNA molecules. The described suppression of endogenous target gene is to use antisense lncRNA to have the function of competing endogenous target RNA, insert strong promoter on target gene through CRISPR Cas9 gene editing technology, synthesize a large amount of antisense lncRNA complementary to target gene, realize In situ cis-competitive repression of endogenous target gene expression.

[0017] 2. Construction of targeting vector pCD Cas9-gRNA1-pU6-gRNA2

[0018] Two gRNAs with pU6 and pH1 promoters were cloned into the targeting vector, and Pme I and Not I restriction sites were inserted behind the Cas9 nuclear l...

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Abstract

The invention discloses a method for inhibiting target gene expression by antisense lncRNA-mediated cis-regulation. The method for inhibiting gene expression by antisense lncRNA-mediated cis-regulation is characterized in that by utilizing the characteristic that the lncRNA has the effect of competing endogenous target RNA, a strong promoter is inserted on a target gene by taking RNA as a genome positioning tool through a CRISPR Cas9 gene editing method, and a lot of antisense lncRNA complementary with the target gene is synthesized to realize in situ cis-competitive inhibition of expression of the endogenous target gene. The method comprises the following steps: firstly, constructing a targeting vector and a homologous template strand donor vector, simultaneously transfecting the constructed vectors into host cells, performing target cloning by puromycin and ganciclovir co-screening, and finally, performing detection and functional research on expression of the target gene.

Description

technical field [0001] The present invention belongs to the field of molecular biology. More specifically, antisense lncRNA has the function of competing with endogenous target RNA, and a strong promoter is reversely inserted on the target gene through CRISPR Cas9 gene editing method, and a large amount of antisense lncRNA complementary to the target gene is synthesized to achieve in situ Cis competition inhibits the expression of endogenous target genes, which is a method for antisense lncRNA to mediate cis regulation to inhibit the expression of target genes. Background technique [0002] Abnormally high expression of certain genes in various human tumors stimulates cells to grow, proliferate or migrate, and finally leads to cancer or metastasis of cells. These highly expressed genes can be used as targets for treating tumors. How to precisely target and inhibit the expression of these genes is the key. The current means of gene suppression is mainly post-transcriptional...

Claims

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Application Information

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IPC IPC(8): C12N15/90
CPCC12N15/902
Inventor 文雪胡继繁孙京男崔久嵬李赵志
Owner JILIN UNIV
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