HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Data An erratum has been published All Versions of this Article: 2006-0634v1 25/6/1521    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liew, C.-G. Articles by Andrews, P. W. Search for Related Content PubMed PubMed Citation Articles by Liew, C.-G. Articles by Andrews, P. W.

EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES

Transient and Stable Transgene Expression in Human Embryonic Stem Cells

Centre for Stem Cell Biology, Department of Biomedical Science, Western Bank, University of Sheffield, Sheffield, United Kingdom

Key Words. Transfection • Human embryonic stem cell • Gene silencing • Promoter

Correspondence: Peter W. Andrews, Ph.D., Centre for Stem Cell Biology, Department of Biomedical Science, Western Bank, University of Sheffield, Sheffield S10 2TN, United Kingdom. Telephone: +44-(0)114-222-4173; Fax: +44-(0)114-222-2399; e-mail: p.w.andrews{at}sheffield.ac.uk

Received on October 5, 2006; accepted for publication on February 27, 2007.

First published online in STEM CELLS EXPRESS  March 22, 2007.


Plasmid vectors remain a valuable yet capricious tool for the genetic manipulation of human embryonic stem (hES) cells. We have compared the efficacy of four promoters to mediate transient and stable transfection in hES and human embryonal carcinoma cell lines with the reporter enhanced green fluorescent protein (eGFP). In transient assays, the two mammalian promoters, UbiquitinC and Rosa26 (pUbiC and pR26), the human cytomegalovirus major immediate early promoter (HCMV-MIE; pCMV), and the HCMV-MIE/chicken β-actin/rabbit β-globin hybrid promoter (pCAGG) gave variable results that depended upon the cell line transfected but in an unpredictable way: each promoter supported strong transient expression in at least one cell line. The results for stable transfection were generally at variance with the transient assays. In each case, transgene silencing was quite marked, most notably with the pCMV, with which no eGFP-positive clones were obtained. An exception was the pCAG vector, in which the CAGG composite promoter is linked to the polyoma virus mutant enhancer PyF101; stable eGFP-positive transfectants were obtained, and these clones retained eGFP expression for over 120 passages, even in the absence of selection. However, if the PyF101 elements were removed, the resulting transfectants were also subjected to progressive gene silencing. Thus, the choice of promoter is critical for determining the desired effect of transgene expression in hES cells. Our data also demonstrate that pUbiC, pR26, pCAGG, and pCAG are more superior to the pCMV for generation of stable transfectants in hES cells.

Disclosure of potential conflicts of interest is found at the end of this article.

This article has been cited by other articles:

				Z.-W. Du, B.-Y. Hu, M. Ayala, B. Sauer, and S.-C. Zhang Cre Recombination-Mediated Cassette Exchange for Building Versatile Transgenic Human Embryonic Stem Cells Lines Stem Cells, May 1, 2009; 27(5): 1032 - 1041. [Abstract] [Full Text] [PDF]

				N. J. Harrison, J. Barnes, M. Jones, D. Baker, P. J. Gokhale, and P. W. Andrews CD30 Expression Reveals that Culture Adaptation of Human Embryonic Stem Cells Can Occur Through Differing Routes Stem Cells, May 1, 2009; 27(5): 1057 - 1065. [Abstract] [Full Text] [PDF]

				T. I. Orban, A. Apati, A. Nemeth, N. Varga, V. Krizsik, A. Schamberger, K. Szebenyi, Z. Erdei, G. Varady, E. Karaszi, et al. Applying a "Double-Feature" Promoter to Identify Cardiomyocytes Differentiated from Human Embryonic Stem Cells Following Transposon-Based Gene Delivery Stem Cells, May 1, 2009; 27(5): 1077 - 1087. [Abstract] [Full Text] [PDF]

				G. Zafarana, S. R. Avery, K. Avery, H. D. Moore, and P. W. Andrews Specific Knockdown of OCT4 in Human Embryonic Stem Cells by Inducible Short Hairpin RNA Interference Stem Cells, April 1, 2009; 27(4): 776 - 782. [Abstract] [Full Text] [PDF]

				C. Unger, E. Karner, A. Treschow, B. Stellan, U. Felldin, H. Concha, M. Wendel, O. Hovatta, A. Aints, L. Ahrlund-Richter, et al. Lentiviral-Mediated HoxB4 Expression in Human Embryonic Stem Cells Initiates Early Hematopoiesis in a Dose-Dependent Manner but Does Not Promote Myeloid Differentiation Stem Cells, October 1, 2008; 26(10): 2455 - 2466. [Abstract] [Full Text] [PDF]

				K. A. Hohenstein, A. D. Pyle, J. Y. Chern, L. F. Lock, and P. J. Donovan Nucleofection Mediates High-Efficiency Stable Gene Knockdown and Transgene Expression in Human Embryonic Stem Cells Stem Cells, June 1, 2008; 26(6): 1436 - 1443. [Abstract] [Full Text] [PDF]

				J. R. Smith, S. Maguire, L. A. Davis, M. Alexander, F. Yang, S. Chandran, C. ffrench-Constant, and R. A. Pedersen Robust, Persistent Transgene Expression in Human Embryonic Stem Cells Is Achieved with AAVS1-Targeted Integration Stem Cells, February 1, 2008; 26(2): 496 - 504. [Abstract] [Full Text] [PDF]

				B. Thyagarajan, Y. Liu, S. Shin, U. Lakshmipathy, K. Scheyhing, H. Xue, C. Ellerstrom, R. Strehl, J. Hyllner, M. S. Rao, et al. Creation of Engineered Human Embryonic Stem Cell Lines Using phiC31 Integrase Stem Cells, January 1, 2008; 26(1): 119 - 126. [Abstract] [Full Text] [PDF]