The use of engineered E1A genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines.
Vectors expressing adenovirus 5 E1A or a domain 2 mutant E1A were introduced into CHO-K1 cells in order to transactivate the hCMV-MIE promoter in transient and stable transfections. Expression from the hCMV promoter was efficiently activated by both wild-type and mutant E1A in contrast to other viral promoters such as the SV40 early promoter which are repressed by E1A. E1A genes expressed from a strong promoter were inhibitory to the growth of CHO cells. Nevertheless, by the use of a weaker promoter, it was possible to isolate stably transfected cell lines containing a level of E1A compatible with both continued cell growth and significant transactivation of the hCMV promoter. By this means we have generated cell lines secreting tissue inhibitor of metalloproteinases (TIMP) at levels approaching those previously attained using gene amplification. CHO cell lines constitutively expressing wild-type and mutant E1A genes have been derived which can serve as new host cell lines for transient expression and efficient stable expression without gene amplification.
Cockett
M I
MI
Celltech Ltd, Slough, UK.
Bebbington
C R
CR
Yarranton
G T
GT
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D005822:Genetic Vectors; D006023:Glycoproteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012313:RNA / Q000032:analysis; D019714:Tissue Inhibitor of Metalloproteinases; D015534:Trans-Activators; D014162:Transfection
D006023:Glycoproteins; D019714:Tissue Inhibitor of Metalloproteinases; D015534:Trans-Activators; D012313:RNA; D008666:Metalloendopeptidases