Construction and identification of over-expression lentivirus vector of rat GSK-3 beta (Glycogen Synthase Kinase-3 beta) target gene

Abstract

The invention discloses construction of an over-expression lentivirus vector of a rat GSK-3 beta (Glycogen Synthase Kinase-3 beta) target gene, comprising the following steps of: tagging the GSK-3 beta target gene by using a PCR (Polymerase Chain Reaction) method; performing directional exchange on the GSK-3 beta target gene and an enzyme-cutting linear vector to obtain a product; converting the product into bacterial competent cells; performing bacterial colony PCR identification on grown clones; and sequencing positive clones identified by the PCR. The invention further discloses identification of the over-expression lentivirus vector of the rat GSK-3 beta target gene, comprising the following steps of: transfecting rat WB-F344 (Western Blot F344) cells by the target gene over-expression lentivirus vector, a vacuolating virus vector and physiological saline; after achieving optimal infection multiplicity, observing expression of fluorescent protein; and detecting the expression condition by adopting Western Blot. According to the construction and identification disclosed by the invention, action mechanisms of the GSK-3 beta in organisms and related diseases thereof can be known deeply; pathogenesis of the diseases can be known better; and a foundation for future clinical application can be laid.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and biomedicine, and specifically relates to a method for constructing a rat GSK-3β target gene overexpression lentiviral vector and a method for identifying a rat GSK-3β target gene overexpression lentiviral vector. . Background technique [0002] The full name of GSK-3β is glycogen synthase kinase-3β, GSK-3β, that is, glycogen synthase kinase-3β, is a serine / threonine protein kinase, widely present in various cells, and is a multifunctional expression enzyme. GSK-3β was initially only found to be involved in the storage and metabolism of muscle energy, but with the deepening of research, it was found that this enzyme not only acts on multiple cell signaling pathways such as Wnt, Notch, hedgehog, and insulin signal transduction, but also in cell apoptosis. It plays an important regulatory role in cell death, gene expression, cell proliferation and differentiation, tumor occurrence, i...

AI Technical Summary

Problems solved by technology

Currently commonly used viral vectors include retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, etc., but these vectors have shortcomings, which seriously affect the effectiveness and safety of gene therapy.

The main problem of retroviral vectors widely used in gene therapy is that they cannot efficiently transduce non-dividing cells. Although adenoviral vectors can transduce non-dividing cells, they cannot achieve stable long-term expression of target genes in vivo , and repeated application is likely to cause immune response

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