Cloning Of Fragile Histidine Triad Gene And Identifying It's Expression In Hela Cell

The fragile histidine triad (FHIT) gene is a new candidate of anti-oncogene, and locates on the human chromosome 3pl4.2. In this research, FHIT cDNA, which is 493bp, was cloned by RT-PCR from the liver tissue total RNA, then was inserted into the cloning vector pMD18-T. After selecting the recombined plasmids which the FHIT gene inserted into correctly, we named it pMD18-T-FHIT. We digested the plasmids with restriction endonuclease in order to recover the target fragments. These fragments were ligated into the multiple cloning site (MCS) of the bicistronic expression vector pIRES2-EGFP, co-expression vector pEGFP-C1 and efficient expression vector pcDNA3.1(+). Then the vectors were transfected into HeLa cell with lipofectamine Regent respectively, 48 hours later, we could observe the green fluorescence emitting from the transfected cells under the fluorescence microscope. G418 was used to select the stable transfected cells, then the total DNA was extracted, and the target products of PCR were 493bp-long, this result confirmed that the FHIT gene had been integrated into cell genome steadily. From the result of RT-PCR we concluded that the gene was transcripted into RNA constantly. Further more, after immunocytochemistry in the fixed transfected cells and negative control with rabbit anti-FHIT, via DAB coloring, the transfected cells became brown, however, the negative control was colourless. It meant...