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

被引:0
|
作者
Tabar, Mehdi Sharifi [1 ]
Hesaraki, Mahdi [2 ]
Esfandiari, Fereshteh [2 ]
Samani, Fazel Sahraneshin [2 ]
Vakilian, Haghighat [1 ]
Baharvand, Hossein [2 ]
机构
[1] ACECR, Royan Inst Stem Cell Biol & Technol, Dept Mol Syst Biol, Cell Sci Res Ctr, Tehran, Iran
[2] ACECR, Royan Inst Stem Cell Biol & Technol, Dept Stem Cells & Dev Biol, Cell Sci Res Ctr, Tehran, Iran
关键词
Electroporation; Lipofectamine; Genetic Modification; HOMOLOGOUS RECOMBINATION; FUNCTIONAL-CHARACTERIZATION; TRANSFECTION; NUCLEOFECTION; PROTEIN; CLONING; GENE;
D O I
暂无
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Objective: 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 reports 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. Materials and Methods: In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for transient expression of green fluorescent protein in two genetically different hESC lines, Royan 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 transfection 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. Results: Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation efficiency revealed that the vector concentrations from 20-60 mu g and the density of the subjected cells (5x10(5) and 1x10(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. Conclusion: 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.
引用
收藏
页码:438 / 450
页数:13
相关论文
共 50 条
  • [21] Differentiation of human embryonic stem cells on periodontal ligament fibroblasts in vitro
    Inanc, Buelend
    Elcin, A. Eser
    Uensal, Evrim
    Balos, Koeksal
    Parlar, Ates
    Elcin, Y. Murat
    ARTIFICIAL ORGANS, 2008, 32 (02) : 100 - 109
  • [22] An inducible transgene expression system for regulated phenotypic modification of human embryonic stem cells
    Fu, Ji-Dong
    Jung, Yunjoon
    Chan, Camie W.
    Li, Ronald A.
    STEM CELLS AND DEVELOPMENT, 2008, 17 (02) : 315 - 324
  • [23] An inducible transgene expression system for regulated phenotypic modification of human embryonic stem cells
    Ji-Dong Fu
    Camie WY Chan
    Ronald A Li
    Cell Research, 2008, 18 : S133 - S133
  • [24] Transgene Independent Germ Cell Differentiation From Human Embryonic Stem Cells (hESCs).
    Tran, N. D.
    Lair, D.
    Kissner, M.
    Conti, M.
    Blelloch, R.
    FERTILITY AND STERILITY, 2010, 93 (05) : S22 - S22
  • [25] An inducible transgene expression system for regulated phenotypic modification of human embryonic stem cells
    Fu, Ji-Dong
    Chan, Camie W. Y.
    Li, Ronald A.
    CELL RESEARCH, 2008, 18 (Suppl 1) : S133 - S133
  • [26] Evaluating human embryonic germ cells: Concord and conflict as pluripotent stem cells
    Turnpenny, Lee
    Spalluto, Cosma M.
    Perrett, Rebecca M.
    O'Shea, Marie
    Hanley, Karen Piper
    Cameron, Iain T.
    Wilson, David I.
    Hanley, Neil A.
    STEM CELLS, 2006, 24 (02) : 212 - 220
  • [27] Stable generation of neural precursor cells from human embryonic stem cells
    Lee, Song-ee
    Park, Mira
    Lee, Nayeon
    Chun, Byounggi
    Choi, SeongJun
    Song, Jihwan
    TISSUE ENGINEERING AND REGENERATIVE MEDICINE, 2008, 5 (02) : 248 - 252
  • [28] Effect of a feeder layer composed of mouse embryonic and human foreskin fibroblasts on the proliferation of human embryonic stem cells
    Yang, Hua
    Qiu, Ying
    Zeng, Xianghui
    Ding, Yan
    Zeng, Jianye
    Lu, Kehuan
    Li, Dongsheng
    EXPERIMENTAL AND THERAPEUTIC MEDICINE, 2016, 11 (06) : 2321 - 2328
  • [29] Stable genetic modification of human embryonic stem cells by lentiviral vectors
    Gropp, M
    Itsykson, P
    Singer, O
    Ben-Hur, T
    Reinhartz, E
    Galun, E
    Reubinoff, BE
    MOLECULAR THERAPY, 2003, 7 (02) : 281 - 287
  • [30] FIDELITY OF TARGETED RECOMBINATION IN HUMAN FIBROBLASTS AND MURINE EMBRYONIC STEM-CELLS
    ZHENG, H
    HASTY, P
    BRENNEMAN, MA
    GROMPE, M
    GIBBS, RA
    WILSON, JH
    BRADLEY, A
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (18) : 8067 - 8071