Transient in vivo reporter gene assay for ecdysteroid action in the Bombyx mori silk gland

Abstract

To analyze the molecular mechanisms underlying hormone-regulated gene expression during molt and metamorphosis, we developed a transient reporter gene assay system using the silkworm anterior silk gland. Reporter plasmids were delivered into dissected anterior silk glands by particle bombardment and bombarded glands transplanted into other larvae, to which hormones were then administered. When the green fluorescent protein gene , coupled with the constitutive cytoplasmic actin gene A3 promoter , was introduced into the anterior silk gland, strong green fluorescence was observed a few days later. Bombarded silk glands transplanted into other larvae showed the same morphological changes as intrinsic glands after 20-hydroxyecdysone (20E) alone or 20E plus juvenile hormone (JH) treatment, indicating that the transplanted gland received hormonal signals properly. When a 20E-responsive reporter construct containing four tandemly repeated pal-1 ecdysone response elements upstream from the luciferase gene was delivered into the gland, an approximately 50-fold increase in luciferase activity was detected 30 h after 20E injection. This induction was comparable to that in an ecdysteroid-responsive Bombyx cell line. This in vivo reporter assay system is thus a rapid, effective tool for analyzing gene expression regulated by 20E and probably by JH.