Construction of a Human Liver Carcinoma Cell Line that Stable Expression of HBV with Gene Trap Vector / 中国生物工程杂志

ABSTRACT

To establish a cell model in vitro that stable expressing HBV by integrating HBA1.3 DNA into cell chromosome. The HBV1.3 full-length DNA was obtained by digested pGEM-HBV1.3 plasmid with HindIII and then was linked with PU-21 vector digested by HindIII. This was resulted in generation of a recombined plasmid named PU21-HBV plasmid. The recombined plasmid was introduced into HepG2 cells by electroporation. The transfected cells were screened with G418. The insertion and expression of HBV were identified by X-gal staining, RT-PCR and Southern blot. The result of PU21-HBV plasmid sequence demonstrated that HBV1.3 DNA was linked correctly with PU-21 vector. A series of positive cell colonies were obtained with G418 screening followed transfecting PU21-HBV plasmid into HepG2 cells. The results of Southern blot and RT-PCR exhibit that HBV1.3 DNA had successfully integrated into the chromosomes of HepG2 cells and had functional HBV gene transcription. HBV1.3 DNA was inserted into HepG2 genome and could stable transcript HBV RNA. The stable HBV expression cell line was constructed successfully. There are LoxP sites in the trapping vector PU21. With the Cre enzyme, interesting genes could be excganged into the LoxP sites. Therefore, double stable expression of interesting gene and HBV cell lines could be generated. The cell lines will be useful for further research some target gene function on replication of HBV.