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...

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Bibliographic Details
Published in:British journal of haematology 2000-09, Vol.110 (3), p.674-681
Main Authors: McIvor, Zoe J., Heyworth, Clare M., Johnson, Barbra A., Pearson, Stella, Fiegler, Heike, Hampson, Lynn, Dexter, T. Michael, Cross, Michael A.
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Cell Culture Techniques
Cell Differentiation - genetics
Cell differentiation, maturation, development, hematopoiesis
Cell Line
Cell physiology
Cell Survival - genetics
Colony-Forming Units Assay
Electroporation
FDCP‐mix cells
Fundamental and applied biological sciences. Psychology
Gene Expression
Genes, Regulator
Genetic Vectors - genetics
green fluorescent protein
Green Fluorescent Proteins
haemopoiesis
Hematology
Hematopoietic Stem Cells - metabolism
Humans
Luminescent Proteins - genetics
Mice
Molecular and cellular biology
Receptors, Granulocyte-Macrophage Colony-Stimulating Factor - analysis
transient transfection
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Summary: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.
ISSN:0007-1048
1365-2141
DOI:10.1046/j.1365-2141.2000.02214.x