疾病动物模型,全基因敲除小鼠 z-bio 2021-03-08 1106

　　Whole gene knockout is to replace the target gene with a foreign DNA sequence (in most cases, the Neomycin resistance gene used for drug screening), so as to achieve the purpose of inactivating the target gene.

　　The construction of targeting plasmids for complete gene knockout is relatively simple. Generally, there is a positive screening gene (such as NeoR, Hygromycin Resistant Gene) and a negative screening gene (such as TK, DTA). The positive selection gene is located in the middle of the two homology arms, and the negative selection gene is generally located outside of one homology arm. The positive selection gene is similar to the resistance genes such as ampicillin on the plasmid in molecular biology experiments. Before transfecting mouse embryonic stem cells, the targeting vector is generally linearized, and then the targeting vector is introduced into the mouse embryonic stem cells by electrotransformation using an electroporator. The linear targeting vector integrates into the chromosome. In the presence of antibiotics (such as G418), only embryonic stem cells recombined with the targeting vector on their chromosomes can survive and proliferate. In the case of homologous recombination, the negative selection gene will be lost, resulting in inability to express. If it is inserted randomly, the negative screening gene outside the homology arm will generally be retained.

　　Traditionally, the negative screening gene is generally used for the thymidine kinase gene TK from HSV. This kinase converts Gancyclovir into a nucleoside analogue and integrates into the newly synthesized DNA strand during the process of genome replication, resulting in DNA Copying stops. Therefore, Gancyclovir can inhibit the proliferation of embryonic stem cells randomly inserted into the targeting vector. Since Gancyclovir often affects the germline transmission of chimeric mice, TK gene is rarely used as a negative screening gene in targeting vectors. The most commonly used is DTA (Diphtheria Toxin Subinit A, diphtheria toxin A subunit). In the case of random insertion, if DTA is expressed, DTA can directly kill the cells, and there is no need to add Gancyclovirs for negative screening.