TRANSFECTION OF AVIAN LMH-2A HEPATOMA-CELLS WITH CATIONIC LIPIDS

LMH-2A is an estrogen-responsive avian hepatoma cell line whose suscep tibility to cationic-lipid-mediated transfection is poorly described. 3 beta[N-N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DCC) requi res a one-step synthesis, and can be used to formulate transfection-gr ade liposomes when combined with dioleoylphosphatidyl-ethanolamine (DO PE) 1/1 (wt/wt). Luciferase activities in LMH-2A cells were 8.5-fold a nd 87.5-fold greater than those in HepG2 and FTO2B cells, respectively , following DCC-liposome-mediated transfection with a reporter consist ing of the human cytomegalovirus immediate-early promoter (CMV), joine d to Photinus pyralis luciferase (L) cDNA, designated pCMVL. Using pCM VL, -dimethyl-2,3-bis(9-octadecenyloxy)-propanaminimun bromide) (BMOP) /DOPE 1/1 (wt/wt), at a 7.5:1 ratio with DNA, produced luciferase acti vities that were 2.9-fold higher than those of DCC-liposomes, at an op timal 10:1 lipid:DNA ratio. At optimal lipid:DNA ratios, commercially available liposomes, Transfectam(R), Lipofectamine(R), and Lipofectin( R), produced luciferase activities that were 1.39, 1.03, and 0.47-fold those of DCC-liposomes. The effect of 0, 10, 100, or 500 nM/L 17 beta -estradiol on the expression of pCMVL and a second luciferase reporter containing the -593/+48 promoter region of the estrogen-responsive av ian apo VLDL-II gene, designated pApoL, was tested in cells cultured i n the presence or absence of 10% chicken serum. The CMV promoter suppo rted a high level of expression in LMH-2A cells that was unaffected by serum alone, but was weakly responsive to estrogen. Estrogen response s of both reporters reached a plateau at 10 nM/L. Estrogen increased t he expression of pApoL 24-fold and 79-fold in the absence and presence of serum, respectively. The -593/+48 region of the apo VLDL-II promot er may not contain previously reported negative insulin response eleme nts, but chicken serum contains factors that enhance estrogen responsi veness of this region.