Construction and Identification of pSIREN-HIF-1α/shRNA Expression Vector / 昆明医科大学学报

ABSTRACT

Objective To construct and identity pSIREN-HIF-1α/shRNA expression vector in order to make foundation of gene therapy for further exploration of RNA interference to nasopharyngeal darcinoma. Methods According to HIF-1αcDNA gene sequence in the gene bank (NM_001530/NM_181054), a pair of 60 nt oligonucleotides each containing the sites of restriction endonuclease at both ends,were designed and synthesized by Reynolds design principles. Oligonucleotides were annealed and ligated with linedrized RNAi-Ready pSIREN-RetroQ-ZsGreen.Transfected into JM109, the recombinants were finally sequenced and identified by 1%agarose gel electrophoresis. Results The size of the target gene fragment amplified by PCR was 470 bp and in accordance with the expected result.pSIREN-HIF-1α was successfully constructed and identitfied by 1%agarose gel electrophoresis.Sequence analysis of inserted fragment revealed the same sequence as synthesized shRNA Oligonucleotides. Conclusion pSIREN-HIF-1α /shRNA expression vector has been successfully constructed, and can make the foundation of research using liposome packaging transfectiing nasopharyngeal darcinoma cell for the next step .