An efficient binary system for gene expression in the silkworm, Bombyx mori, using GAL4 variants

Abstract

A binary gene expression system using the yeast GAL4 DNA-binding protein and the upstream activating sequence (UAS) of galactose-driven yeast genes is an established and powerful tool for the analysis of gene function. However, in the domesticated silkworm, Bombyx mori, this system has been limited in its utility by the relatively low transcriptional activation activity of GAL4 and by its toxicity. In this study, we investigated the potential of several established GAL4 variants (GAL4Δ, GAL4VP16, GAL4VPmad2, GAL4VPmad3, and GAL4NFκB) and of two new GAL4 variants, GAL4Rel and GAL4Relish, which contain the transcription-activating regions of the BmRel and BmRelish genes, respectively, to improve the utility of the GAL4/UAS system in B. mori. We generated constructs containing these GAL4 variants under the control of constitutive or inducible promoters and investigated their transcription-activating activity in cultured B. mori cells and embryos and in transgenic silkworms. GAL4VP16 and GAL4NFκB exhibited high transactivation activity but appeared to be toxic when used as transgenes under the control of a constitutive promoter. Similarly, GAL4VPmad2 and GAL4VPmad3 exhibited higher transactivation activity than GAL4, combined with strong toxicity. The transcription-activating activity of GAL4Δ was about twice that of GAL4. The two new GAL4 variants, GAL4Rel and GAL4Relish, were less active than GAL4. Using GAL4VP16 and GAL4NFκB constructs, we have developed a very efficient GAL4/UAS binary gene expression system for use in cultured B. mori cells and embryos and in transgenic silkworms.

A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori

“…In the latter case, ectopic ser1 was observed in some GAL4/ϩ PSGs (Fig. 1D), which might result from the transcriptional perturbation; in the silkworm, GAL4 frequently exhibits a toxic effect (32)(33)(34). We conclude that ser1 is regulated by Antp because it was expressed in all Ayfib-GAL4/UAS-Antp PSGs and was also induced by hs-GAL4 (Fig.…”

SID-1 protein of Caenorhabditis elegans mediates uptake of dsRNA into Bombyx cells

“…This is a chimeric protein harboring the DNA binding domain of yeast Gal4 and the transcriptional activation domain of mouse nuclear factor-kB (Ogura et al, 2009). To normalize transfection efficiencies, we also transfected pRL-ie1 (Kobayashi et al, 2011), which expresses Renilla reniformis luciferase (Rluc) under control of the baculovirus ie-1 promoter (Jarvis et al, 1990), together with the plasmids described above. To express SID-1 adequately under the GAL4NFkB/UAS system, the cells were incubated at 27 C in serum-containing medium for 2 days.…”

“…The transfection procedure was performed using HilyMax reagent (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. The following plasmids were used to assess the effects of the expression of sid-1: pBac[UAS-sid-1, 3xP3-egfp], pBac [hsp70B-Fluc, 3xP3-egfp], and pBac[A3-gal4NFkB, 3xP3-DsRed2] (Kobayashi et al, 2011). Gal4NFkB is one of the Gal4 variants with a stronger transcriptional activation function than the native Gal4 protein.…”

Krüppel Homolog 1 Inhibits Insect Metamorphosis via Direct Transcriptional Repression of Broad-Complex, a Pupal Specifier Gene

“…p65AD_BmKr-h1 expression vector in which BmKr-h1 was fused with the activation domain of p65 (p65AD), a subunit of NF-B (41). Through this modification, if p65AD_BmKr-h1 bound to the BmBR-C_P dist region, the luciferase reporter downstream of the BmBR-C_P dist region would be forcibly activated by p65AD.…”