Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

Objective: genetic modification of human embryonic stem cells (hescs) is critical fortheir extensive use as a fundamental tool for cell therapy and basic research. despitethe fact that various methods such as lipofection and electroporation have been appliedto transfer the gene of interest (goi) into the target cell line, however, there are few reportsthat compare all parameters, which influence transfection efficiency. in this study,we examine all parameters that affect the efficiency of electroporation and lipofection fortransient and long-term gene expression in three different cell lines to introduce the bestmethod and determinant factor.materials and methods: in this experimental study, both electroporation and lipofectionapproaches were employed for genetic modification. pcag-egfp was applied for transientexpression of green fluorescent protein in two genetically different hesc lines, royanh5 (xx) and royan h6 (xy), as well as human foreskin fibroblasts (hff). for long-termegfp expression vasa and olig2 promoters (germ cell and motoneuron specific genes,respectively), were isolated and subsequently cloned into a pblumar5 plasmid backboneto drive egfp expression. flow cytometry analysis was performed two days after transfectionto determine transient expression efficiency. differentiation of drug resistant hesccolonies toward primordial germ cells (pgcs) was conducted to confirm stable integrationof the transgene.results: transient and stable expression suggested a variable potential for different celllines against transfection. analysis of parameters that influenced gene transformation efficiencyrevealed that the vector concentrations from 20-60 ?g and the density of the subjectedcells (5×105 and 1×106 cells) were not as effective as the genetic background andvoltage rate. the present data indicated that in contrast to the circular form, the linearizedvector generated more distinctive drug resistant colonies.conclusion: electroporation was an efficient tool for genetic engineering of hescscompared to the chemical method. the genetic background of the subjected cell linefor transfection seemed to be a fundamental factor in each gene delivery method. foreach cell line, optimum voltage rate should be calculated as it has been shown to playa crucial role in cell death and rate of gene delivery .