Building and application of chronic virus transformation vector with CMV-CBh double promoters

Abstract

The invention relates to building and application of a chronic virus transformation vector with CMV-CBh double promoters. The chronic virus vector pLenti-CMV-3FLAG-EGFP-tCBh-mCherry-T2A-Puro is obtained by replacing PGK in the pLenti-CMV-3FLAG-EGFP-tCBh-mCherry-T2A-Puro with tCBh fragments obtained by performing PCR (Polymerase Chain Reaction) amplification. The tCBh promoter is used for driving expression of mCherry gene and Puromycin resistance gene, and cells for stabilizing over-expression of target gene can be screened by using Puromycin; the mCherry gene is beneficial for judging the virus packaging efficiency and the infection efficiency, and is also beneficial for stabilizing the screening of the cells. Transfection fluorescence observation proves that the chronic virus vector expression system with the double promoters is successfully built, and a basic vector is provided for further researching target genes of interest.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to the construction and application of a lentivirus CMV-CBh double promoter transformation vector. Background technique [0002] Bidirectional promoter: A DNA sequence located between two adjacent genes that are transcribed in opposite directions. [0003] Bidirectional promoters are widely distributed in eukaryotic genomes. Most bidirectional promoters lack TATA boxes, but have high GC content and abundant CpG islands. Bidirectional promoters initiate transcription bidirectionally, and will bidirectionally transcribe Genes play a role in the regulation of co-expression and stable expression. [0004] In the process of genetically improving organisms, it may be necessary to transfer more than one exogenous gene into a specific organism, and these exogenous genes need to be expressed under the control of their corresponding promoter sequences. Because they are used...

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Problems solved by technology

[0004] In the process of genetically improving organisms, it may be necessary to transfer more than one exogenous gene into a specific organism, and these exogenous genes need to be expressed under the control of their corresponding promoter sequences. Because they are used for genetic improvement The number of promoters is limited, so a specific promoter or a promoter with a similar sequence is often repeatedly used when transferring multiple genes into an organism. Although this can achieve the desire to transfer multiple genes at the same time, this vector On the other hand, because the introduced promoter sequences are homologous, even if there is only 90 bp sequence homology between the two promoters, the introduction into the organism will cause gene expression "co-suppression" phenomenon, resulting in gene transcription Silence, this is the difficulty and challenge to be faced in transgenic technology.

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