Transient and Stable Transgene Expression in Human Embryonic Stem Cells
Article first published online: 22 MAR 2007
Copyright © 2007 AlphaMed Press
Volume 25, Issue 6, pages 1521–1528, June 2007
How to Cite
Liew, C.-G., Draper, J. S., Walsh, J., Moore, H. and Andrews, P. W. (2007), Transient and Stable Transgene Expression in Human Embryonic Stem Cells. STEM CELLS, 25: 1521–1528. doi: 10.1634/stemcells.2006-0634
- Issue published online: 2 JAN 2009
- Article first published online: 22 MAR 2007
- Manuscript Accepted: 27 FEB 2007
- Manuscript Received: 5 OCT 2006
- Human embryonic stem cell;
- Gene silencing;
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.