Sharifi Tabar Mehdi, Hesaraki Mahdi, Esfandiari Fereshteh, Sahraneshin Samani Fazel, Vakilian Haghighat, Baharvand Hossein
Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Cell J. 2015 Fall;17(3):438-50. doi: 10.22074/cellj.2015.5. Epub 2015 Oct 7.
Genetic modification of human embryonic stem cells (hESCs) is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest (GOI) into the target cell line, however, there are few re- ports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor.
In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for tran- sient expression of green fluorescent protein in two genetically different hESC lines, Roy- an H5 (XX) and Royan H6 (XY), as well as human foreskin fibroblasts (hFF). For long-term EGFP expression VASA and OLIG2 promoters (germ cell and motoneuron specific genes, respectively), were isolated and subsequently cloned into a pBluMAR5 plasmid backbone to drive EGFP expression. Flow cytometry analysis was performed two days after trans- fection to determine transient expression efficiency. Differentiation of drug resistant hESC colonies toward primordial germ cells (PGCs) was conducted to confirm stable integration of the transgene.
Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation ef- ficiency revealed that the vector concentrations from 20-60 μg and the density of the sub- jected cells (5×10(5)and 1×10(6)cells) were not as effective as the genetic background and voltage rate. The present data indicated that in contrast to the circular form, the linearized vector generated more distinctive drug resistant colonies.
Electroporation was an efficient tool for genetic engineering of hESCs compared to the chemical method. The genetic background of the subjected cell line for transfection seemed to be a fundamental factor in each gene delivery method. For each cell line, optimum voltage rate should be calculated as it has been shown to play a crucial role in cell death and rate of gene delivery.
人类胚胎干细胞（hESCs）的基因改造对于其作为细胞治疗和基础研究的基本工具的广泛应用至关重要。尽管诸如脂质转染和电穿孔等各种方法已被用于将目的基因（GOI）导入靶细胞系，然而，很少有报告比较所有影响转染效率的参数。在本研究中，我们研究了影响电穿孔和脂质转染效率的所有参数，以在三种不同细胞系中实现瞬时和长期基因表达，从而引入最佳方法和决定因素。
在本实验研究中，电穿孔和脂质转染方法均用于基因改造。pCAG-EGFP用于在两种基因不同的hESC系Royan H5（XX）和Royan H6（XY）以及人包皮成纤维细胞（hFF）中瞬时表达绿色荧光蛋白。为了实现长期EGFP表达，分离了VASA和OLIG2启动子（分别为生殖细胞和运动神经元特异性基因），随后将其克隆到pBluMAR5质粒骨架中以驱动EGFP表达。转染两天后进行流式细胞术分析以确定瞬时表达效率。对耐药性hESC集落向原始生殖细胞（PGCs）的分化进行检测以确认转基因的稳定整合。
瞬时和稳定表达表明不同细胞系对转染具有不同的潜力。对影响基因转化效率的参数分析表明，20 - 60μg的载体浓度和受试细胞密度（5×10⁵和1×10⁶个细胞）不如遗传背景和电压率有效。目前的数据表明与环状形式相比，线性化载体产生了更多独特的耐药集落。
与化学方法相比，电穿孔是hESCs基因工程的有效工具。受试转染细胞系的遗传背景似乎是每种基因递送方法的一个基本因素。对于每种细胞系，应计算最佳电压率，因为它已被证明在细胞死亡和基因递送速率中起关键作用。
