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

Research output: Contribution to journalArticle

Abstract

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.

Details

Authors
  • I Hamaguchi
  • N B Woods
  • I Panagopoulos
  • E Andersson
  • H Mikkola
  • C Fahlman
  • R Zufferey
  • L Carlsson
  • D Trono
  • S Karlsson
Organisations
External organisations
  • Skåne University Hospital
  • Lund University
  • University of Geneva
  • Umeå University
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Microbiology in the medical area

Keywords

  • Animals, Cell Differentiation, Gene Expression, Gene Expression Regulation, Viral, Gene Transfer Techniques, Genetic Vectors, Green Fluorescent Proteins, HIV-1/genetics, Hematopoietic Stem Cells/physiology, Humans, Luminescent Proteins/genetics, Mice/embryology, Retroviridae/genetics, Transcription, Genetic, Transduction, Genetic, Transgenes, Vesicular stomatitis Indiana virus/genetics, Virus Integration
Original languageEnglish
Pages (from-to)10778-84
JournalJournal of Virology
Volume74
Issue number22
Publication statusPublished - 2000 Nov
Publication categoryResearch
Peer-reviewedYes