Premium
A transient assay for regulatory gene function in haemopoietic progenitor cells
Author(s) -
McIvor Zoe J.,
Heyworth Clare M.,
Johnson Barbra A.,
Pearson Stella,
Fiegler Heike,
Hampson Lynn,
Dexter T. Michael,
Cross Michael A.
Publication year - 2000
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1046/j.1365-2141.2000.02214.x
Subject(s) - transfection , progenitor cell , biology , microbiology and biotechnology , gene , stem cell , green fluorescent protein , gene expression , cellular differentiation , plasmid , interleukin 3 , genetics , in vitro , antigen presenting cell , cytotoxic t cell
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.