Transfection of human kidney epithelial 293 cells with bacterial cytosine-5-methyltransferase M. Msp 1and the SV40 nuclear localisation sequence of VP1

Abstract

The aim of this study was to con-struct a plasmid that would direct the expres-sion of a bacterial DNA methyltransferase in cultured mammalian cells. It was carried out with a PCR-based strategy for introducing three components' coding sequences into the transfection vector pcDNA 3 (Invitrogen). The gene encoding the bacterial cytosine specific DNA methyltransferase M. Msp1 was first introduced into the general cloning vector pMTL23. Then, the coding sequence for glu-tathione- S-transferase was added into the upstream of human viral nuclear localisation sequences SV40 VPI of the prepared con-struct using the PCR technique. The complete synthetic fusion protein gene was finally introduced into the general purpose transfection vector pcDNA 3 . The plasmid encoded fusion protein was demonstrated by exhibit ing cytosine specific DNA methyltransferase activity by resistance to the cognate restriction enzyme R. Msp1 digestion following transformation into E.coli. Human kidney epithelial 293 cells were transfected with DNA methyltransferase vector using an antibody directed against the glutathione-Stranferase moiety, so that the enzyme was expressed in whole cells. Furthermore, substantial phenotypic changes were observed in the 293 cells following transient transfection. Total protein and genomic DNA samples were prepared from the transfected cells and attempts were made to establish that genomic DNA was methylated at bacterial ( Moroxella spp.) MTase M. Msp1 sites.

The aim of this study was to con-struct a plasmid that would direct the expres-sion of a bacterial DNA methyltransferase in cultured mammalian cells. It was carried out with a PCR-based strategy for introducing three components' coding sequences into the transfection vector pcDNA 3 (Invitrogen). The gene encoding the bacterial cytosine specific DNA methyltransferase M. Msp1 was first introduced into the general cloning vector pMTL23. Then, the coding sequence for glu-tathione- S-transferase was added into the upstream of human viral nuclear localisation sequences SV40 VPI of the prepared con-struct using the PCR technique. The complete synthetic fusion protein gene was finally introduced into the general purpose transfection vector pcDNA 3 . The plasmid encoded fusion protein was demonstrated by exhibit ing cytosine specific DNA methyltransferase activity by resistance to the cognate restriction enzyme R. Msp1 digestion following transformation into E.coli. Human kidney epithelial 293 cells were transfected with DNA methyltransferase vector using an antibody directed against the glutathione-Stranferase moiety, so that the enzyme was expressed in whole cells. Furthermore, substantial phenotypic changes were observed in the 293 cells following transient transfection. Total protein and genomic DNA samples were prepared from the transfected cells and attempts were made to establish that genomic DNA was methylated at bacterial ( Moroxella spp.) MTase M. Msp1 sites.