The Construction of a Mammalian Transfection Vector For Expression of Cytosine-5 Specific DNA Methyltransferase Gene M.Msp1 In Cultured Cells

Authors: Öztürk ÖZDEMİR

Abstract: The expression vectors are designed for expression and purification of normal or recombi-nant gene of interest. There is a wide variety of stable and transferable selectable mammalian expres-sion vectors.The vector pCDM8 and its derivatives, pcDNAI/Ampicilline are widely used for cloning and analyzing of genes in higher eukaryotic cells. The vectors pRC/CMV, pcDNA3 and pRC/RSV are designed for high-level expression of recombinant genes in mammalian cells. In the present study, we have been able to transfect and express the monospecific bacterial (Moroxcella spp) cytosine-5 DNA methyltransferase (MTase) M.Msp1 gene in cultured cells within a newly con-structed mammalian transfection vector. We have constructed a very efficient, stable or transferable eukaryotic expression vector analogous to the well-known expression system in COS cells. Bacterialcytosine-5 MTase gene with a vertebrate nuclear targeting signal (NLS) SV40 VP1 and mark-er enzyme glutathion-S-transferase (GST) genes were transferred into the eukaryotic shuttle vector pcDNA3 using a PCR based method. This novel plasmid which contains a strong cytomegalovirus and T7 promoters and the SV40 origin of replication for autonomous replication in mammalian cells was-called pOZT4. Human kidney epithelial 293 and CHO cells were transfected with pOZT4 which was encoding the fusion gene and it was established that the genomic DNA of both cells were methylated at the CCGG sites by the active enzyme.

Keywords: Vector pOZT4, Bacterial methyl transferase M.Msp1, Mammalian cell transfection.

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