Allele double knockout targeting vector system and construction method thereof

Abstract

The invention relates to an allele double knockout targeting vector system and a construction method thereof. The system of the invention consists of two complementary vectors, namely pGT-V1 and pGT-V2, wherein each vector contains two in phase LoxP elements which contain a positive selection marker gene Neo / GFP and Hyg / RFP respectively; and the outside of each vector contains a negative selection marker gene TK. Meanwhile, two 8-basic group multiple cloning sites are designed and arranged between the two LoxP elements and the negative selection marker for the insertion of the homology arm. By adopting the constructed targeting vector system of the invention, two complementary targeting vectors are cotransfected into the recipient cell; through the selection of drug and fluorescent double-selection marker, the genetic modification or knockout of the two alleles of the target gene can be realized once; and the interaction of the transfected Cre enzyme and the LoxP elements can be utilized to remove the selection marker gene integrated with the genome, the time of obtaining the homozygous knockout target cell can be shortened, the safety of the transgenic animal can be increased and a valuable technology platform is provided to develop the animal transgenic researches.

Description

[0001] Technical Field: Genetic Engineering technical background: [0002] Research on transgenic animals is now a hot topic in the scientific community. Through transgenic technology, people can break the isolation between species to obtain economic animals with excellent traits, obtain various biological materials and medicinal proteins through the production of bioreactors, break immune rejection for organ transplantation, and establish animal models of various human diseases As well as analysis to study specific gene function. [0003] However, the existing transgenic technology still has drawbacks. The biggest problem is the position effect caused by the random insertion of foreign genes, that is, the random insertion of foreign genes into genes related to important life activities of cells causes their abnormal expression. In addition, if the gene of interest is randomly inserted into chromatin to generate higher-order structures, or inserted into a heterochromatin regi...

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Problems solved by technology

However, this method takes a long time, and there are very few surviving cells under the pressure of high concentrations of antibiotics, and if the single-copy gene knockout cells develop resistance to the maximum concentration of antibiotics, the strategy will fail

Furthermore, it is difficult to determine whether the cell death is caused by homozygous knockout of key genes or by antibiotic intolerance, which affects the analysis of experimental results

Due to the genetic development characteristics of mice, homozygous gene knockout mice can be obtained through the chimera approach, but in large animals, this process takes a long time and requires a lot of work

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