ISSN:
0006-3592
Keywords:
metabolic engineering
;
CHO cell
;
E2F-1
;
serum-free cell culture
;
two-dimensional electrophoresis of proteins
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
Engineering of the cell cycle can be an effective means for bypassing growth factor requirements of animal cells. Cloned human E2F-1 from Nalm 6 cells was subcloned into pRc/CMV and transfected into Chinese hamster ovary (CHO) cells. Ten stable transfectant clones isolated from cells cultured under neomycin-resistance selection pressure all expressed significantly higher amounts of E2F-1 than control cells as determined by Western analysis. Confocal immunofluorescent microscopy and Southern analysis of several clones also provided evidence for the expression of cloned E2F-1 in these cells. CHO K1:E2F-1 cells are able to proliferate on well-defined serum- and protein-free basal medium and exhibit an S-phase extended by 65% compared to CHO K1 cells mitogenically stimulated by basic fibroblast growth factor (bFGF). Two-dimensional electrophoresis of the intracellular proteins of E2F-1 clones shows an increase in 236 gene products compared to CHO K1 control cells, further verifying a functional regulatory role of cloned E2F-1 in CHO cells. Among these upregulated species is the cell cycle regulatory protein, cyclin A, which has already been shown to be regulated by E2F-1 in human fibroblasts. Overexpression of cloned E2F-1 in CHO cells is a potentially useful new strategy for bypassing serum requirements in mammalian cell culture. Furthermore, such cell cycle control stimulus-protein pattern response data can contribute to a clearer understanding of complex multigene networks involved in mammalian cell cycle regulation. © 1996 John Wiley & Sons, Inc.
Additional Material:
7 Ill.
Type of Medium:
Electronic Resource
