A transient assay for regulatory gene function in haemopoietic progenitor cells.
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.
McIvor
Z J
ZJ
Laboratory of Molecular Medicine, IZKF University of Leipzig, Leipzig, Germany. zoem@medizin.uni-leipzig.de
Heyworth
C M
CM
Johnson
B A
BA
Pearson
S
S
Fiegler
H
H
Hampson
L
L
Dexter
T M
TM
Cross
M A
MA
D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D003114:Colony-Forming Units Assay; D018274:Electroporation; D015870:Gene Expression; D005809:Genes, Regulator; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000032:analysis
D008164:Luminescent Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D049452:Green Fluorescent Proteins