CMV promotor activity during ES cell differentiation: potential insight into embryonic stem cell differentiation.
The activity of the P(CMV IE) promoter was studied during the differentiation of ES cells into neurons. In order to do this, stable embryonic stem (ES) cell lines that express enhanced green fluorescent protein (EGFP) under the control of P(CMV IE) were created and these ES cells were differentiated by aggregation of cells in the presence of retinoic acid (RA). Based on our observations that the activity of P(CMV IE) was highest in undifferentiated cells, and that cell-cell interaction and addition of RA that lead to enhanced cell proliferation also increased expression from P(CMV IE), we hypothesized that the activity of P(CMV IE) was positively regulated in cycling cells. However, when analysis was done at the single cell level it was found that BrdU label and EGFP expression were not correlated. EGFP expression was found to be down-regulated in many cells that were BrdU positive and conversely there were significant numbers of BrdU negative cells that were EGFP positive. Further, P(CMV IE) activity was not observed in cells that were nestin positive or in differentiated neurons, but P(CMV IE) was active in cells with a fibroblast-like morphology. Finally, several proteins present in undifferentiated ES cells were found to bind to regulatory regions of P(CMV IE). These were absent when cells were aggregated in the presence of RA. The above results have implications for expression of transgenes in ES cells as well as providing new insight into the mechanism of lineage restriction.
Bagchi
Bandita
B
National Brain Research Center, Manesar, Gurgaon 122050, India.
Kumar
Manoj
M
Mani
Shyamala
S
D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D002449:Cell Aggregation; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics*; D003587:Cytomegalovirus / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007381:Intermediate Filament Proteins / Q000235:genetics; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics; D064231:Nestin; D009474:Neurons / Q000166:cytology; D011401:Promoter Regions, Genetic; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology; D019076:Transgenes / Q000235:genetics; D014212:Tretinoin / Q000494:pharmacology
D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014212:Tretinoin; D001973:Bromodeoxyuridine