Objective    To clone platelet-derived growth factor A chain (PDGF-A) gene sequence

construct pLXSN-PDGF-A plasmid and express it in porcine bone marrow derived mesenchymal stem cells (bMSCs).     Methods    cDNA fragment encoding human PDGF-A gene were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with the total mRNA isolated from human hepatoma cell line SMC-7721 as template. The PCR amplified fragment of PDGF-A gene was first cloned into pMD18-T vector

and then subcloned into the recombinant retroviral vector pLXSN to construct pLXSN/PDGF-A after seqencing. Subsequently

plasmid DNA of pLXSN/PDGF-A was transfected into packaging cell line PA317

and positive cell clones with higher titer were screened. bMSCs were infected with cell-free supernatant of the highest titer clone transfected with pLXSN/PDGF-A. The transfection and expression of PDGF-A in bMSCs were identified by RT-PCR

Southern blot hybridization and enzyme-linked immunosorbent assay (ELISA). Light microscopy

methyl thiazolyl tetrazolium  (MTT) assay

and induced bone and fat formation were employed to examine the growth and proliferation of bMSCs transfected with PDGF-A and their multilineage differentiation as stem cells.      Results    Full-length cDNA sequence encoding human PDGF-A gene was successfully cloned

and recombinant retroviral vector of pLXSN/PDGF-A is constructed. Stable expression of PDGF-A in porcine bMSCs was obtained with transfection of retroviral supernatant from pLXSN/PDGF-A clone. bMSCs after PDGF-A transfection were grown and proliferated well

and still possessed the trait of stem cells.     Conclusion    cDNA sequence encoding human PDGF-A gene is successfully cloned into pLXSN vector. Stable expression of PDGF-A gene in bMSCs is implemented by retroviral transfection. bMSCs transfected with PDGF-A still possesses the trait of stem cells.