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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 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)...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Royan Institute
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601864/ https://www.ncbi.nlm.nih.gov/pubmed/26464815 |
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author | Sharifi Tabar, Mehdi Hesaraki, Mahdi Esfandiari, Fereshteh Sahraneshin Samani, Fazel Vakilian, Haghighat Baharvand, Hossein |
author_facet | Sharifi Tabar, Mehdi Hesaraki, Mahdi Esfandiari, Fereshteh Sahraneshin Samani, Fazel Vakilian, Haghighat Baharvand, Hossein |
author_sort | Sharifi Tabar, Mehdi |
collection | PubMed |
description | 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 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. MATERIALS AND METHODS: 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. RESULTS: 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. 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. |
format | Online Article Text |
id | pubmed-4601864 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Royan Institute |
record_format | MEDLINE/PubMed |
spelling | pubmed-46018642015-10-13 Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells Sharifi Tabar, Mehdi Hesaraki, Mahdi Esfandiari, Fereshteh Sahraneshin Samani, Fazel Vakilian, Haghighat Baharvand, Hossein Cell J Original Article 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 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. MATERIALS AND METHODS: 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. RESULTS: 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. 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. Royan Institute 2015 2015-10-07 /pmc/articles/PMC4601864/ /pubmed/26464815 Text en Any use, distribution, reproduction or abstract of this publication in any medium, with the exception of commercial purposes, is permitted provided the original work is properly cited http://creativecommons.org/licenses/by/2.5/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Article Sharifi Tabar, Mehdi Hesaraki, Mahdi Esfandiari, Fereshteh Sahraneshin Samani, Fazel Vakilian, Haghighat Baharvand, Hossein Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells |
title | Evaluating Electroporation and Lipofectamine Approaches
for Transient and Stable Transgene Expressions in
Human Fibroblasts and Embryonic Stem Cells |
title_full | Evaluating Electroporation and Lipofectamine Approaches
for Transient and Stable Transgene Expressions in
Human Fibroblasts and Embryonic Stem Cells |
title_fullStr | Evaluating Electroporation and Lipofectamine Approaches
for Transient and Stable Transgene Expressions in
Human Fibroblasts and Embryonic Stem Cells |
title_full_unstemmed | Evaluating Electroporation and Lipofectamine Approaches
for Transient and Stable Transgene Expressions in
Human Fibroblasts and Embryonic Stem Cells |
title_short | Evaluating Electroporation and Lipofectamine Approaches
for Transient and Stable Transgene Expressions in
Human Fibroblasts and Embryonic Stem Cells |
title_sort | evaluating electroporation and lipofectamine approaches
for transient and stable transgene expressions in
human fibroblasts and embryonic stem cells |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601864/ https://www.ncbi.nlm.nih.gov/pubmed/26464815 |
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