Methylated and Unmethylated pDNA Delivery Comparison in Mammalian Cells

In this study, the differences in delivery of methylated and unmethylated prokaryotic

DNA in mammalian cells was investigated. 3 plasmids, DH5-α, ER2925 and

GM272 were extracted and transformed from E. coli bacteria. DH5-α is the regular

methylated plasmid, however,ER2925 and GM272 lack Dam and Dcm enzymes which

methylate adenine and internal cytosine in prokaryotes respectively, hence they are

unmethylated. The 3 plasmids were delivered via different delivery vectors in two

cell lines, UMUC3 and MDA-MB-231 which are human bladder cancer cell line and

human triple negative breast cancer cell line, respectively.

Luciferase and BCA assay were carried out to quantify transgene expression to

compare the efficacy of gene delivery in three aforementioned plasmids. In addition,

hydrodynamic diameter and zeta potential was measured for all delivery vectors, to

correlate with other transgene expression data. The results show that methylated

plasmid has significantly higher transgene expression in mammalian cell lines. This

can be either a result of smaller size and more positive zeta potential that the methylated

plasmid had, or a result of having Dam and Dcm enzymes which enhance binding

of DNA and transcription factors and enhance gene expression. Having smaller size

and more positive zeta potential was proven to be the case for the methylated plasmid

in this study. However the latter hypothesis should be investigated furthermore.