DNA transfection in the ecdysteroid-responsive GV1 cell line from the tobacco hornworm, Manduca sexta.
The embryonic cell line, GV1, from Manduca sexta was transiently transfected with DNA constructs of the Drosophila hsp70 promoter fused to either a beta-galactosidase (pXH70ZT) or a chloramphenicol acetyl transferase (HSP-CAT-1) reporter gene using lipofectin. Optimal cell density, DNA:lipofectin ratio, and time of incubation were varied to determine the optimal conditions: 2 x 10(5) cells/ml, 1:3, and 5 h. Under these conditions, the transfection efficiency was about 40%. Heat inducibility of two hsp70 constructs was compared. The HSP-CAT-1, containing 1127 bp of upstream sequence, was more sensitive to heat shock than that of pXH70ZT, containing only 194 bp of upstream sequence. Thus, the 1127 bp hsp70 promoter appears to be a better inducible promoter in these cells. A 2 kb fragment of the proximal promoter region of the MHR3 gene containing a putative ecdysone response element was shown to be responsive to 20-hydroxyecdysone after its transfection into these cells.
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Department of Zoology, University of Washington, Seattle 98195-1800, USA.
Riddiford
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D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000235:genetics*; D004330:Drosophila / Q000235:genetics; D004441:Ecdysterone / Q000494:pharmacology*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D006358:Hot Temperature; D018402:Manduca; D010714:Phosphatidylethanolamines; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D018840:HSP70 Heat-Shock Proteins; D010714:Phosphatidylethanolamines; D011993:Recombinant Fusion Proteins; D004441:Ecdysterone; C020888:1,2-dielaidoylphosphatidylethanolamine; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase