A transient assay for regulatory gene function in haemopoietic progenitor cells.
McIvor, Zoe J Heyworth, Clare M Johnson, Barbra A Pearson, Stella Fiegler, Heike Hampson, Lynne Dexter, T Michael Cross, Michael A
McIvor, Zoe J
Heyworth, Clare M
Johnson, Barbra A
Pearson, Stella
Fiegler, Heike
Hampson, Lynne
Dexter, T Michael
Cross, Michael A
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Abstract
This work aimed to provide a means of assaying directly the effects of transient expression of introduced genes on the survival, proliferation, lineage commitment and differentiation of haemopoietic progenitor cells. For this purpose, we have developed a system that allows isolation of productively transfected, mulitipotent haemopoietic cells within a few hours of the introduction of test genes. We have shown that FDCP-mix cells productively transfected with expression plasmids encoding green fluorescent protein (GFP) differentiate normally and retain colony-forming potential. We constructed an expression vector consisting of a bicistronic cassette in which a GFP marker gene and a test gene are driven from the same promoter. The vector design has been optimized for co-expression and the test gene was shown to be biologically active. The expression profile from a transiently transfected template under different growth conditions reveals that active expression continues for at least 2 d after transfection. The transient transfection of FDCP-mix cells with the vectors described provides a powerful tool for analysis of the immediate early effects of test gene overexpression during haemopoietic differentiation.
Description
Date
2000-09
Publisher
Collections
Keywords
Haematopoietic Stem Cells
Type
Article
Citation
A transient assay for regulatory gene function in haemopoietic progenitor cells. 2000, 110 (3):674-81 Br. J. Haematol.