The Basic Research Of 293t Cells Transfected By HHCN2 Gene To Greate Biologic Pacemakers

Background:Hyperpolarization-activated, cyclic nucleotide-gated(HCN) current plays an important role in regulating heart spontaneous pulsation.Objective:To observe target gene expression on mRNA and protein level of 293t cells transfected with hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) gene recombinant plasmid.Methods:1,Plasmid CMV-hHCN2-3xHA-IRES-EGFP including full length hHCN2 gene was transfected into 293t cells by Liposome Lipofectamine 2000.2,Expression of hHCN2 mRNA was detected qualitatively and quantificationally by reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (RT-PCR).3,Expression of HCN2 protein was examined with Western-blot qualitatively.Results:1,Plasmid CMV-hHCN2-3xHA-IRES-EGFP including full length HCN2 gene was successfully transfected into 293t cells by Liposome Lipofectamine 2000. 2,Reverse transcription polymerase chain reaction detected the highly visible specific DNA bands at 100bp-250bp, which was the same as hHCN2 gene carried by plasmid. HHCN2 mRNA expression between experimental and control groups was statistically significant difference (P=0.006), which detected by real-time quantitative polymerase chain reactions. Whereas the hHCN2 mRNA expression of experimental group is more than the control group, hHCN2 mRNA expression of experimental group was 2 to 4 times of the control group.3,Expression of hHCN2 channel protein has been examined with Western-blot qualitatively, and the expression of transfected group was significantly higher than no treatment group and control group. Conclusion:Plasmid CMV-hHCN2-3xHA-IRES-EGFP including full length HCN2 gene is successfully transfected into 293t cells. MRNA and protein of hHCN2 have been expressed in 293t cells.