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

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...

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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 hairpin RNA (shRNA) in vivo, which can be processed to form molecules similar to siRNAs, which can trigger the RNAi process and can be transfected with viral vectors. This method solves the problem of not being able to permanently inhibit gene expression , can also use inducible promoters such as DOX to induce Tet-on system to control shRNA expression, but their common problem is off-target effect, siRNA may bind to non-target genes and cause non-target gene silencing

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