[Expression of Fas genes transduced into colorectal cancer cells]

To construct colorectal cancer cells expressing exogenous Fas gene and observe the expression level of its mRNA and protein before and after transduction.

Fas cDNA was inserted into the multiple cloning site of the expression vector pBK-CMV with molecular cloning technique, and the resultant recombinant plasmid was transduced into colorectal cancer LoVo cells via lipofectamine. G418 was utilized to screen the positive clones containing the recombinant plasmid, where Fas mRNA and protein expression was determined with Western blotting and dot blotting.

pBK-CMV Fas cDNA plasmid was successfully constructed. The transduced colorectal cancer cells were screened by G418 and a resistant cell line (LoVo Fas cells) was obtained. Fas expression was detected in both transduced and non-transducted cell lines, but the expression level of both Fas mRNA and protein was much higher in the former, which showed lowered proliferation rate and lengthened doubling time and logarithm growth period than the non-transducted cells, but the difference was not significant. Treatment of the transduced cells with Fas antibody produced significant difference (P<0.05), manifested by apparently inhibited cell growth.

LoVo cells normally has only very low expression level of Fas gene, while transduction with pBK-Fas cDNA can enhance the efficiency of Fas mRNA and protein expressions. Fas antibody significantly inhibits the growth and proliferation of in vitro cultured Fas-expressing LoVo cells.