Evidence for the induction of cuticle proteins by 20‐hydroxyecdysone in two established insect cell lines

Abstract

We have studied two different established insect cell lines capable of synthesizing chitin, UM-BGE-4 from the cockroach and BRL-AC-2 from the boll weevil, for their ability to produce putative cuticle proteins. Mouse polyclonal antibodies were raised against the abdominal cuticle proteins extracted from the cuticle of nymphal and adult stage cockroaches, and larval, pupal, and adult stage cotton boll weevils. O n Western blots, these antibodies detected several cross-reacting proteins in untreated control cells and a variety of induced proteins in cells first exposed to a double pulse of the molting hormone, 20-hydroxyecdysone (a low concentration followed by a higher concentration of hormone). The majority of these hormone induced proteins recognized by the anti-cuticle protein antibodies were found to correspond in size to specific proteins observed in abdominal cuticle extracts. Electron microscopy utilizing colloidal gold to identify antibody binding sites indicated the antigenic proteins were present in the extracellular material found on the surface of the cultured cockroach and boll weevil cells. The evidence presented suggests that these cell lines can produce cuticle proteins. 01992 WiIey-Liss, Inc.

Transfection of cultured cells of the cotton boll weevil, Anthonomus grandis, with a heat‐shock‐promoter‐chloramphenicol‐acetyltransferase construct*

“…However, receptor molecules are found in the cytoplasm and nucleus of responsive insect cells (Dinan etal., 1990), and they apparently bind to specific sequences on the genomic DNA after ligand activation (or binding) (Bidmon & Sliter, 1990). Therefore, ecdysteroid conjugation to polylysine might increase the frequency of stable integration of DNA into ecdysteroid responsive cells (such as the BRL-AG-2 weevil cell line; Stiles et al, 1992;Stiles & Newman, 1992) and this could be tested using a plasmid incorporating a selectable marker gene (such as antibiotic resistance; Rio & Rubin, 1985;van der Straten eta/., 1988).…”

“…Multiple pulse electroporation (3 pulses, 175 V, 0.56 ms duration, 30pg ml-'DNA) did give a weak CAT gene expression and approximately 70% cell death. The low cell survival after electroporation could be a serious drawback in the adaptation of this procedure for stable transformation of insect cells due to the relatively low cloning efficiency (very sensitive to low cell densities) of some insect cell lines (Marks, 1980;Stiles et a/., 1992). Certainly, electroporation was substantially less effective (based on CAT gene expression or amount of DNA used) than Lipofectin for transfecting the BRL-AG-3C cells.…”

“…With the recent development of cell lines from the cotton boll weevil, Anthonornus grandis Boheman (Stiles et a/., 1992) we now have a system for testing genetic constructs for eventual transformation of this and perhaps other coleopteran insects. In this study we have utilized the Drosophila 70,000 molecular weight (MW) heat shock protein (hsp 70) promoter combined with a bacterial chloramphenicol acetyltransferase (CAT) reporter gene (Di Nocera & Dawid, 1983) in a transient gene expression system to compare the transfection efficiency of several cationic carrier systems (polylysine, polybrene and Lipofectin) with that of electroporation of the BRL-AG9C cell line.…”