Construction Of PIRES2-AcGFP1-CD Eukaryotic Expression Plasmid And Its Expression In Bone Marrow Mesenchymal Stem Cells (MSCs)

Cytosine deaminase (CD) is an enzyme found in a variety of bacteria and fungi where iscapable of deaminating cytosine to uracil. The existence of CD in a prokaryotic cell rendersthe cell selectively sensitive to 5-FC. Selective expression of CD within neoplastic cells whencombined with systemic 5-FC administration may result in locally high concentration of 5-FUwhile minimizing the systemic 5-FU toxicity.Currently, mesenchymal stem cells (MSCs) is a hot interest in the stem cell's area. MSCscan transdifferentiate into multiple cell lineages. The character of MSCs, for example themultipotention, easy isolation, cultivation and proliferation, low immunogenicity, easy betransfected and expression exotic gene and so on, makes them ideal engineering cells in celland gene therapies.In this study, MSCs of rabbit is used as engineering cells to express the CD. CD gene isconstructed with fluorescent recombinatant eukaryotic expression plasmid, and then theplasmid is transfected into MSCs for gene therapy of central nervous system diseases.According to the open reading frame of the cytosine deaminase gene, the specificoilgonucleotide primers are designed. Restriction enzyme XhoI and BamHI are designed atthe 5' end and the 3' end, respectively. The cytosine deaminase gene is obtained from E.coliJM109 genome DNA by Polymerase Chain Reaction (PCR). After harvested and purified, thefragment is cloned into pMD19-T vector, and then these are transferred into DH5αcompetentcells. Positive clones are selected by LB plates with Ampicillin and proliferated. By digestionwith restriction enzyme BamHI/XhoI and DNA sequence analysis showed that CD gene wasidentical with the published sequence. The pIRES2-AcGFP1-CD plasmid is constructed andthen identified by restriction enzyme BamHI/XhoI digestion analysis. Throughliposome-mediated method the plasmid is transfected into the marrow mesenchymal stemcells. The transfected marrow mesenchymal stem cells display green fluorescence underfluorescence microscope. To transfection MSCs using pIRES2-AcGFP1-CD plasmidsconducted by lipofectamine 2000 liposomes is a simple, efficient, easy and successfultransfection method. It is a fairly ideal gene method, and very likely that bone marrowmesenchymal stem cells to become the ideal vector in gene thrapy.