Abstract:

BACKGROUND: Hyperpolarization-activated cyclic nucleotide-gated (HCN) currently plays an important role in regulating heart spontaneous pulsation.
OBJECTIVE: Target gene expression was observed at mRNA and protein level in 293T cells transfected with human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) gene recombinant plasmid.
METHODS: Plasmid CMV-hHCN2-3xHA-IRES-EGFP including full length hHCN2 gene was transfected into 293T cells by Liposome Lipofectamine 2000. Expression of hHCN2 mRNA was detected by reverse transcription polymerase chain reaction and real-time quantitative polymerase chain reaction respectively. Expression of HCN2 protein was examined by western blot method.
RESULTS AND CONCLUSION: Plasmid CMV-hHCN2-3xHA-IRES-EGFP including full length HCN2 gene was successfully transfected into 293T cells by Liposome Lipofectamine 2000. Reverse transcription polymerase chain reaction detected the highly visible specific DNA bands at 100-250 bp, which was the same as hHCN2 gene carried by plasmid. Reverse transcription-polymerase chain reaction and western blot method results showed that hHCN2 gene was highly expressed at mRNA and protein levels. These findings suggest that hHCN2 gene was expressed at the nucleic acid and protein levels in 293T cells transfected by plasmid of CMV-hHCN2-3xHA-the IRES-EGFP.