Construction and application of a mouse HPRT targeting vector with Cre recombinase recognition site lox66
- VernacularTitle:基于Cre重组酶体系的小鼠HPRT定点整合打靶载体的构建及其应用
- Author:
Jingmin ZHENG
;
Jian LI
;
Hua YANG
;
Jiliang FU
;
- Publication Type:Journal Article
- Keywords:
gene targeting;
embryonic stem cell;
genes,HPRT;
Cre recombinase
- From:
Academic Journal of Second Military Medical University
1985;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To study the construction and application of a vector with Cre recombinase recognition site lox66 for mouse HPRT gene targeting in embryonic stem(ES) cells. Methods: Using the HPRT genomic DNA fragment and synthesized oligonucleotides, pSP HPRT lox66 Neo was designed and constructed as a replacement vector by common molecular cloning techniques. After the linearized pSP HPRT lox66 Neo DNA was electroporated into ES cells, and transfected cells were cultured in G418/6 TG drug selection medium. The recombination efficiency of this vector was tested. Results: The main components of pSP HPRT lox66 Neo were a positive selection gene Neo, lox66, long and short homologous fragments of mouse HPRT gene and plasmid backbone. Twenty ES cell clones with HPRT gene inactivated were obtained. Conclusion: An effective replacement vector with Cre recombinase recognition site lox66 is constructed and applied to HPRT gene targeting in ES cells.
