ABSTRACT
AIM: To construct a knock-out vector for ?~(maj) & ?~(min) gene of ?-globin gene and establish embryonic stem cell lines by gene knock-out in ES level for generating gene knock-out animal model. METHODS: Two homologous sequences 1.7 kb and 7.0 kb were inserted into basic plasmic vector pNTK, which contains positive and negative selection system of the neomycin resistance (Neo) and herpes simplex virus thymidine kinase (TK) resistant gene to construct a gene knock-out recombinant plasmic vector nominated ?-pNTK, which was certified by polymerase chain reaction, enzyme digested and DNA sequencing. The ?-pNTK that contains two homologous sequence was linearized with Sal I and introduced into the D3 line of ES cells by electroporation. Positive cloning of ES cells was selected with positive and negative selection system of G418 600 mg/L and 2.5 mol/L Gancyclovir for four weeks. ES cells clone were picked, explanded and certified by polymerase chain reaction, Southern blotting. Examination of the undifferentiated state and pluripotential characteristics of the cell lines were made with the alkaline phosphatase (AKP) staining and embryoid bodies formation test and teratomas formation in nude mice. RESULTS: A knock-out recombinant plasmic vector for ? maj & ? min gene of ?-globin gene, ?-pNTK, was constructed in C129 mouse, which contains 8.7 kb length inserted homologous sequence and deleted most part of the ?~(maj) & ?~(min) gene. 8 strains of positive ES cells cloning were obtained, all of them were testified positive with polymerase chain reaction and Southern blotting. The results of AKP staining, embryoid bodies formation in vitro and teratomas formation in nude mice showed undifferentiated state and pluripotential characteristic of cell line. CONCLUSIONS: A knock-out recombinant plasmic vector of ?~(maj) & ?~(min) of ?-globin gene in C129 mouse was constructed, in which a knock-out ?~(maj) & ?~(min) gene of ?-globin gene ES-D3 cell line was established by electroporation, characterization of these cells show a prospect utility in producing gene knock-out mice in blastocysts stages.
