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Journal of Virology, November 2000, p. 10778-10784, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Lentivirus Vector Gene Expression during ES Cell-Derived Hematopoietic Development In Vitro

Isao Hamaguchi,1 Niels-Bjarne Woods,1 Ioannis Panagopoulos,1 Elisabet Andersson,1,dagger Hanna Mikkola,1,Dagger Cecilia Fahlman,1 Romain Zufferey,3 Leif Carlsson,2 Didier Trono,3 and Stefan Karlsson1,*

Molecular Medicine and Gene Therapy, Department of Medicine, Lund University Hospital, Lund,1 and Department of Microbiology, Umeå University, Umeå,2 Sweden, and Department of Genetics and Microbiology, University of Geneva, Geneva, Switzerland3

Received 15 May 2000/Accepted 19 August 2000

The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular 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 hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified 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 hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system 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.

* Corresponding author. Mailing address: Molecular Medicine and Gene Therapy, Lund University, WNC, Sölvegatan 17, 223 62 Lund, Sweden. Phone: 46 46 222 05 77. Fax: 46 46 222 05 78. E-mail: Stefan.Karlsson{at}molmed.lu.se.

dagger Present address: Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.

Dagger Present address: Children's Hospital, Division of Hematology/Oncology, Boston, Mass.

Journal of Virology, November 2000, p. 10778-10784, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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