Cell and Tissue Journal

Abstract Aim: In this study the efficiency of lentiviral gene transfer and cytomegalovirus promoter mediated overexpression has been investigated. Furthermore, the effect of small molecule-mediated inhibition of epigenetic pathways on gene overexpression has been studied. Materials and Methods: Mouse ES cells were transduced with different doses of lentiviruses  harboring Green Fluorescent Protein (GFP) and the percentage of GFP expressing cells was quantified with flowcytometery. The persistence of GFP expression was assessed after 8 days of transduction. Using embryonic stem (ES) cells transduced with a lentiviral vector harboring pancreatic and duodenal 1 (Pdx1) gene, we studied the influence of 5-azacytidin (5-AZA), DZNep, and BIX01294 small molecules on the gene expression system. Results: Lentiviral mediated gene transfer to ES cells with 10 and 20 multiplicities of infection (MOI) resulted in more than 90 percent transgenesis. However, the expression of transgene showed a dramatic decrease during 8 days of post-transduction. Treatment with 5-AZA leads to a 2.5 folds increase in the transgene expression in a permissive culture medium. DZNep also elevated the transgene expression up to 26.5 and 5.9 folds in pluripotency and permissive media respectively. The expression of endogenous Pdx1 gene also increased following DZNep treated. Conclusion: Lentiviral transduction with MOIs more than 10, is an efficient method for transgenesis of mouse ES cells. However, co-application of this method along with the CMV promoter leads to inactivation of the gene expression in long term. DZNep treatment results in reactivation of transgene expression but also can influence the expression of endogenous genes.