Establishment of A549 cell line with stable expression of HIF1α mediated by lentiviral vector / 重庆医学

ABSTRACT

Objective To establish the A549 cell line with stable expression of HIF1α by using lentiviral vector system.Methods Primers were designed and synthesized with human HIF1α gene coding sequence by the National Center of Biotechnogical Information(NCBI) as the template.HIF1α was amplified by PCR.The HIF1α fragment recycled by enzyme digestion was recombined with prepared lentiviral vector HBLV-RFP-Puro.The recombinant plasmid was identified by PCR and gene sequencing.The recombinant plasmid and the auxiliary plasmid were co-transfect into 293T cell.After filtration and concentration of packaged virus,the viral titer was detected by using the dilution counting method.The prepared lentivirus was infected A549 cells.The drug screening was adopted to stabilize the transfected cell line.The transfection effect was detected and observed by fluorescence microscope and Western blotting.Results The HIF1α fragment amplified by PCR was successfully verified and the recombinant plasmid was successfully constructed by PCR and gene sequencing identification.High-titer LV-HIF1α was obtained by successful package.After LV-HIF1α infecting A549 cells,the cells showed the red fluorescence by fluorescence microscope.The expression level of HIF1α in the LV-HIF1α group was significant higher than that in the control group by Western blot.Conclusion The 549 cell line with HIF1α stable expression mediated by lentivirus is constructed successfully.