ABSTRACT
Objective To develop a new method to induce the gene transfection in high efficiency for eukaryotic cells in vitro. Methods Four kinds of p14ARF gene primarily deleted human carcinoma cell line including H460,A549,U251,and PC-3 were transfected with the human p14ARF expression vector (pCI-neo-p14ARF) by using the new nonliposomal transfection reagent Fugene 6. The efficiency of gene transfer was determined by screening the cells in G418. Results After 21 days' selection, G418-resistant clones were shown in all the transfected plate. PCR product of p14ARF gene was positive in all the G418-resistant clones. Cytotoxicity of Fugene 6 was detected. The cell proliferation activity was not affected when it was cultured in a high dose of Fugene 6. Conclusion These results demonstrate that Fugene 6 is a rapid, feasible, reproducible, and noncytotoxic gene transfection approach for eukaryotic expression vector in vitro.
ABSTRACT
p16 gene was a tumor supressor gene found recently. The p16 protein is a negative regulator of cell proliferation . Loss of normal p16 function is associated with the development of neoplasms. To detect antitumorigenic effect of p16 on human lung adenocarcinoma cells, we cloned a p16 cDNA into the pcDNA3 vector at the sites of BamHl and Xho I to gain a p16 gene recombinant expression vector plasmid. We then transferred the p16 gene recombinant plasmid into human lung adenocarcinoma cell line (NCI-H460) by electroporation method. After G418 selection we assessed cell growth properties and cell cycle pattern by flow cytometry to G418-resistant clones of NCI-H460-pl6 and NCI-H460-vect respectively. The results show that human p16 gene could suppress the phenotype of NCI-H460 cell line and p16 gene therapy plays a positive role in human lung adenocarcinoma treatment.