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ARS Home » Midwest Area » Columbia, Missouri » Biological Control of Insects Research » Research » Publications at this Location » Publication #99327
Title: EXPRESSION OF THE GREEN FLUORESCENT PROTEIN CARRIED BY AUTOGRAPHA CALIFORNICA MULTIPLE NUCLEAR POLYHEDROSIS VIRUS IN INSECT CELL LINES

Author
item Grasela, James
item McIntosh, Arthur
item Goodman, Cynthia
item WILSON, LOUISE - OXFORD BROOKES UNIV, UK
item KING, LINDA - OXFORD BROOKES UNIV UK

Submitted to: In Vitro Cellular And Developmental Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/16/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: The expression of the green fluorescent protein has been used as an important indicator of cell and virus interaction. However, little information is available on its expression in insect cells. In this study experiments were conducted to determine which of 14 insect cell lines is the best virus producer when inoculated with a virus carrying the gene that texpresses a green light-emitting protein. A cell line is made by taking cells from live insects and growing them in a liquid medium. Of the 14 cell lines tested, all allowed virus growth and produced green light-emitting protein to varying degrees except one of the cell lines from the tobacco budworm, corn earworm and silkworm which did not show expression of the green light-emitting protein. Cabbage looper cells produced the largest mean percent number of green light-emitting cells in growth medium after addition of virus. Virus production at 120 h after addition of virus was highest in beet armyworm cells followed by the tobacco budworm cells. Thi information has important implications as a means to facilitate further understanding of the infectious process of baculoviruses as biological control agents of insects as well as determining the best insect cell lines for the production of green light-emitting protein. The results of this study provides further information important to the scientific community and industry on the susceptibility of 14 insect cell lines to a modified baculovirus containing the green light-emitting protein gene and its expression.

Technical Abstract: A recombinant AcMNPV containing the green fluorescent protein gene (gfp) under the polyhedrin promoter was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in nine continuous insect cell lines including Heliothis virescens (BCIRL-HV- AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), ,Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), its clone Sf9 Spodoptera exigua (Se-E1; Se-E5), Bombyx mori (BMN), and five cell line clones of BCIRL-HV-AM1. Of the 14 cell lines tested, all were permissive with varying degrees to AcMNPV.GFP except BCIRL-HV-AMCL2, BCIRL-HZ-AM1, and BMN which was nonpermissive to the virus. Except for Se-E1, IPLB-SF21 and 4 of the 5 BCIRL-HV-AM1 clones, all the other cell lines began to express GFP at 48 h postinoculation. Se-E1 in serum-containing medium expressed the GFP at 48 h postinoculation. TN-CL1 cells produced the largest mean percent number of fluorescent cells both in serum-containing (76.6%) and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. ECV production at 120 h postinoculation was highest in Se-E5 cells grown in serum-containing medium (37.8 TCID50/ml x 10**6) followed by BCIRL-HV-AM1 in TC199-MK (33.4 TCID50/ml x 10**6). Only BCIRL-HV-AMCL3 produced any substantial level of ECV at 120 h postinoculation (16.9 TCID50/ml x 10**6). There was no significant correlation between ECV production and the mean percent 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.