Hematopoietic stem cells are widely recognized as attractive targets for gene therapy but current protocols to transduce these cells using recombinant retroviral vectors are inefficient. In order to accurately and noninvasively measure retroviral iransduction of hematopoietic stem cells and conveniently evaluate stability of gene expression in their progeny, the green fluorescent protein (OFF) was explored as a reporter. We first improved sensitivity of detection >100-fold over that achieved previously by expressing an enhanced OFF gene from a derivative of the MSCV (munne stem cell virus) retroviral vector without an internal promoter. Primitive human hematopoietic cells bearing the CD34 surface antigen and lacking lineage differentiation markers (Lin-) were transduced with the new GFP-encoding retroviral vector (termed MGIN) using a clinically applicable supernatant procedure. Under the ex vivo conditions employed, >75% of the target cells retained a CD34+Lin- phenotype after 4-5 days in culture, of which -35% expressed a high level of GFP detectable by both FACS analysis and fluorescence microscopy. When cultured for colony-forming unit-granulocyte/macrophage and burst-forming unitcry throid progenitors (2 weeks) as well as for more immature cobblestone area-forming cells (4-5 weeks), GFP fluorescence was readily detected in situ, indicating that GFP expression was stable and was not detrimental to the differentiative potential of the transduced CD34+Lin- cell population. We conclude that GFP is excellent as a vital marker to quantify retroviral-mediated gene transfer efficiency in human hematopoietic stem cells and monitor gene expression during their subsequent cell lineage determinations. This methodology should facilitate the optimization of retroviral gene delivery technology for the purpose of hematopoietic cell gene therapy.
|Original language||English (US)|
|Number of pages||1|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology
- Cancer Research