A bicistronic co-expression gene transfer body and its preparation method

Abstract

The invention discloses a bicistronic co-expression gene transfer body and a preparation method. The gene sequence of the gene transfer body is SEQ ID NO: 17. The gene transfer body comprises bovine matrix attachment regions (MARs), an RNA polymerase promoter III (U6), short hairpin RNA (shRAN) targeting the third exon of bovine myostatin (MSTN), a CMV promoter, a bovine follistatin (FSTN) and an SV40polyA signal region from the five prime end to the three prime end in order. The gene transfer body provided by the invention doesn't contain a vector backbone sequence and is a clean and safe gene transfer; and the gene transfer body combines the MAR with a knock-down vector for the first time and can effectively enhance the knock-down effect of the vector. Besides, the gene transfer body achieves the purpose of conducting knock-down and over-expression simultaneously by independently starting double promoters, and provides new ideas and paths for improving the characteristics (such as economic characteristics) of double-gene / multi-gene control.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering in the field of biotechnology, and relates to a bicistronic co-expression gene transfer body and a preparation method. Background technique [0002] High-efficiency expression of foreign genes in transgenic animals must rely on good expression vectors. There are many factors that affect the high-efficiency expression of foreign genes, such as promoters, methylation, gene structure, insertion sites, regulatory sequences (enhancers, insulators, nuclear matrix binding regions), etc. [0003] The main function of MSTN is to negatively regulate the growth and development of muscle cells. Although the MSTN gene is quite conserved during evolution, it is not necessary for the survival and reproduction of animals. In MSTN knockout or knockdown mice, no abnormalities were found other than muscular development. In this way, the effect of knocking down the gene on animal muscle development and...

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Problems solved by technology

[0010] In recent years, the gene expression cassette (including only the promoter, coding region and terminator) that removes the backbone sequence of the plasmid vector has been used as a gene transfer body, also known as clean DNA transformation (clean DNA transformation n), which mainly transforms plants through a gene gun. It has been successfully applied in the transformation of rice, cotton, wheat, and grape, and it has also been successfully used in melon by using the pollen tube introduction method. It is rare to see the construction and application in transgenic animals, especially mammals, and there are few reports on RNA interference vectors. However, there is almost no research on the construction of gene expression cassettes based on the combination of MAR sequences and RNA interference.

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