CAJ | 학술논문

目的　建立一种介导体外真核细胞高效率基因转染的新方法。方法　采用一种新的非脂质体基因转染试剂Fugene 6，以人p14ARF蛋白表达载体（pCI-neo-p14ARF）为外源DNA，转染存在p14ARF基因原发缺失的人H460，A549，U251和PC-3共4个癌细胞系，通过G418筛选21　d确定转染效率。结果　经转染的4个细胞系培养孔中均长出了抗G418细胞克隆，而且这些细胞克隆的p14ARF基因PCR产物均为阳性，细胞毒性分析发现高浓度Fugene 6作用下各细胞的增殖活力未受影响。结论　Fugene 6为一简便快速、易操作、重复性好且无细胞毒性的体外真核表达载体基因转染新系统。
목적　건립일충개도체외진핵세포고효솔기인전염적신방법。방법　채용일충신적비지질체기인전염시제Fugene 6，이인p14ARF단백표체재체（pCI-neo-p14ARF）위외원DNA，전염존재p14ARF기인원발결실적인H460，A549，U251화PC-3공4개암세포계，통과G418사선21　d학정전염효솔。결과　경전염적4개세포계배양공중균장출료항G418세포극륭，이차저사세포극륭적p14ARF기인PCR산물균위양성，세포독성분석발현고농도Fugene 6작용하각세포적증식활력미수영향。결론　Fugene 6위일간편쾌속、역조작、중복성호차무세포독성적체외진핵표체재체기인전염신계통。
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.