Host cis -Mediated Extinction of a Retrovirus Permissive for Expression in Embryonal Stem Cells during Differentiation

Abstract

The use of retroviral vectors for gene transfer into animals has been severely hampered by the lack of provirus transcription in the early embryo and embryonic stem (ES) cells. This primary block in provirus expression is maintained in differentiated cells by acis-acting mechanism that is not well characterized. Retroviral vectors based on the murine embryonal stem cell virus (MESV), which overcome the transcriptional block in ES cells, were constructed to investigate this secondary mechanism. These vectors transferred G418 resistance to ES cells with the same efficiency as to fibroblasts, but overall transcript levels were greatly reduced. A mosaic but stable expression pattern was observed when single cells from G418-resistant clones were replated in G418 or assayed for expression of LacZ or interleukin-3. The expression levels in independent clones were variable and correlated inversely with methylation. However, a second, more pronounced, block to transcription was found upon differentiation induction. Differentiation of the infected ES cells to cells permissive for retroviral expression resulted in repression and complete extinction of provirus expression. Extinction was not accompanied by increased levels of methylation. Provirus expression is thus regulated by two independentcis-acting mechanisms: (i) partial repression in the undifferentiated state, accompanied by increased methylation but compatible with long-term, low expression of retroviral genes, and (ii) total repression and extinction during early stages of differentiation, apparently independent of changes in methylation. These results indicate a time window early during the transition from an undifferentiated to a differentiated stage in which provirus expression is silenced. The mechanisms are presently unknown, but elucidation of these events will have an important impact on vector development for targeting stem cells and for gene therapy.

Expression of the LIM-homeobox gene LH2 generates immortalized Steel factor-dependent multipotent hematopoietic precursors

“…All populations analysed contained the MSCV-derived 1.3 kb Neo-transcript. These results confirm the silencing of LTR-driven expression in ES cells upon differentiation (Laker et al, 1998). Semi-quantitative RT-PCR assays revealed that both cells within HPC colonies and the HPC line expressed similar levels of LH2 ( Figure 6B, HPC col., HPC LQ respectively).…”

A Simple and Efficient System for Regulating Gene Expression in Human Pluripotent Stem Cells and Derivatives

“…Hence, when a high level of transgene expression is sought, including expression in glia, lentivirus infection may be an option. Nevertheless, random insertion comes with another drawback, that is, uneven expression among cell populations as observed in this study as well as by others [18,19,22,24]. This is likely due to varied degrees of silencing depending on insertion sites.…”

“…Efforts have been made to produce transgenic hPSC lines with regulatable gene expression [1,23,25,26]. While expression of transgenes (often GFP) can be regulated in stem cells and their differentiated progenies at an early stage, more than often transgenes are no longer expressed and regulatable in mature cells, including neurons [18,19,22,24]. In that regard, this study demonstrates unequivocally that transgene expression in hPSC-differentiated progenies, including neurons, can be regulated by DOX in a dose-dependent manner.…”

Versatile multi-transgene expression using improved BAC TG-EMBED toolkit, novel BAC episomes, and BAC-MAGIC

“…Moreover, foreign sequences by themselves are targets for epigenetic silencing (16–19), and transgene concatamers can induce the formation of heterochromatin (20, 21). Together these transgene silencing mechanisms result in unpredictable transgene expression levels that do not correlate with copy number and are unstable with long-term culture or changes in the cell physiological or differentiated state (22–24).…”