Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in A Promoter-Dependent Manner

Abstract

Lentiviruses have been increasingly used for genetic modification of human cells including embryonic stem (ES) cells. Using four ubiquitous promoters-cytomegalovirus (CMV), cytomegalovirus immediate-early enhancer/chicken β-actin hybrid (CAG), phosphoglycerate kinase (PGK), and human elongation factor-1α (EF1α)-in a lentiviral vector to drive the expression of the enhanced green fluorescent protein (EGFP) gene in human ES cells and mouse ES cells, we determined the extent of EGFP suppression by assessing the percentage of cells that were transduced with the EGFP gene but did not fluoresce green. A much higher level of transgene suppression was observed in human ES cells as compared to mouse ES cells. The suppression was also highly promoter dependent, leading to inactivation of more than 95% of the EGFP genes under the CMV or CAG promoter while only 55% under the PGK promoter. No promoter-dependent suppression was observed in transient transfection of human ES cells. Thus, the common phenomenon of poor transgene expression in human ES cells may be caused mainly by suppression of the transgene right after transduction and integration. Cautions should be taken to choose the optimal promoter when lentiviruses are used for genetic modification of human ES cells.

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…Furthermore, it is well known that a considerable amount of integrated vectors become silent [159] , and this effect seems to be dependent on the promoter chosen to drive the ectopic expression of the gene [160,161] . These observations could be related to ubiquitous RNA guided-DNA methylation pathway mechanism in mammalian cells aimed at controlling endogenous retroviruses.…”

Reprogramming Adipose Tissue-Derived Mesenchymal Stem Cells into Pluripotent Stem Cells by a Mutant Adeno-Associated Viral Vector

“…Such retroviral transgene silencing may result from the activation of potent repressor factors or the reduction of certain activating factors after directly reprogramming somatic cells to pluripotent states (Hotta and Ellis, 2008). In addition, CMV promoter activity has been shown to be progressively silenced in pluripotent stem cells (Xia et al, 2007;Meilinger et al, 2009). In rAAV2.3m-mediated cell reprogramming, whether transgene silencing resulted from the activation of certain endogenous potent repressor or from the reduction of CMV promoter activity in rAAV-iPS cells remains elusive.…”

Human stem cells for modeling neurological disorders: Accelerating the drug discovery pipeline

“…Despite the potential of gene targeting in human ES cells, progress has been hindered by poor transfection and cloning efficiencies, with a handful of accessible cell lines seemingly amenable to manipulation [Xia et al, 2007;Braam et al, 2008]. While for the most part achieving efficient gene delivery remains a problem (i.e., >80% transduction efficiency), transient [Braam et al, 2008] stable [Liew et al, 2007] and conditional [Vieyra and Goodell, 2007] expression systems are evolving, together with more compatible methods of human ES cell culture.…”