Expression And Localization Of PAM14 In Transfected Cell

Objective PAM14 plays an important role in transcription, so we want to clone PAM14 (protein associated with MRG, 14kd) gene into pCDGFP vector and express this vector in appropriate mammalian cell lines to investigate the expression and localization of the product of PAM14 gene.Methods The full length cDNA fragment of PAM14 in pACT2- PAM14 vector was used as template to be amplified with PCR. PCR product and pCDGFP vector were digested by appropriate restriction enzymes separatly, and ligated into pCDGFP-PAM14 construct, which was confirmed by DNA sequencing. The expression of pCDGFP-PAM14 was detected in HEK 293T cells with western-blot and COS7 cells with fluorescence microscope.Results Analysis of restriction enzymes hydrolysis and DNA sequencing demonstrated that the expression vector inserted with PAM14 gene was constructed in frame. This expression vector can express PAM14-GFP protein at significant level in HEK 293T cells, and the PAM14- GFP protein was found to be distributed in the nuclei of COS7 cells, and some in the cytoplasm.Conclusions The expression vector pCDGFP-PAM14 was constructed correctly. The vector pCDGFP-PAM14 can express efficiently in HEK 293T cells, and distributed mainly in the nuclei of COS7 cells. The results provide the basis for further function studies of PAM14.