Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro

The murine embryonal stem (ES) cell virus (MESV) can express transgenes fro m the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES c ells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunod eficiency virus type 1-based lentivirus vector pseudotyped with the vesicul ar stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoie tic cells in vitro. An oncoretrovirus vector containing the MESV LTR and th e GFP gene was used for comparison. Fluorescence-activated cell sorting ana lysis of transduced CCE ES cells showed 99.8 and 86.7% GPF expressing ES ce lls in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefo re, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to ef ficient transduction of ES cells. Lentivirus vector integration was verifie d in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoi etic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the trans gene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental sy stem can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain of-function experiments during ES cell development in vitro.