DNA TRANSFECTION IN THE ECDYSTEROID-RESPONSIVE GV1 CELL-LINE FROM THETOBACCO HORNWORM, MANDUCA-SEXTA

The embryonic cell line, GVI, from Manduca sexta was transiently trans fected with DNA constructs of the Drosophila hsp70 promoter fused to e ither a beta-galactosidase (pXH70ZT) or a chloramphenicol acetyl trans ferase (HSP-CAT-1) reporter gene using lipofectin. Optimal cell densit y, DNA:lipofectin ratio, and time of incubation were varied to determi ne the optimal conditions: 2 x 10(5) cells/ml, 1:3, and 5 h. Under the se conditions, the transfection efficiency was about 40%. Heat inducib ility of two hsp70 constructs was compared. The HSP-CAT-1, containing 1127 bp of upstream sequence, was more sensitive to heat shock than th at of pXH70ZT. containing only 194 bp of upstream sequence. Thus, the 1127 bp hsp70 promoter appears to be a better inducible promoter in th ese 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 cel ls.