Expression of the green fluorescent protein carried by Autographa californica multiple nucleopolyhedrovirus in insect cell lines

A recombinant AcMNPV containing the green fluorescent protein (gfp) gene un der the polyhedrin promoter (polh) was used to investigate the expression o f the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-H V-AM1), Helicoverpa tea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1 ), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/ AMCY-Se-E1 and BCIRL-AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The suscept ibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascerta ined by calculating the mean percentage number of green light-emitting cell s as well as by TCID50 titration of extracellular virus with fluorescence a s a sign of infection. Of the 14 cell lines tested, all were permissive wit h varying degrees to AcMNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, bo th grown in serum-containing medium, and BMN, grown in serum-free medium, w hich were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF2 1, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIR L-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GF P at 72 h postinoculation. By contrast, in BCIRL/ AMCY-Se-E1 in serum-conta ining medium (Ex-Cell 401 + 10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest me an percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM 1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracelluar virus (E CV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cel ls grown in Ex-Cell 401 + 10% FBS (37.8 x 10(6) TCID50/ml) followed by BCIR L-HV-AM1 in TC199-MK (33.4 x 10(6) TCID50/ml). Only the BCIRL-HV-AMCL3 clon e produced any substantial level of ECV at 120 h postinoculation (16.9 x 10 (6) TCID50/ mi). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisio ns as to what other cell lines are available for in vitro studies of the gf p gene.