An efficient binary system for gene expression in the silkworm, Bombyx mori, using GAL4 variants.
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.
Transgenic Silkworm Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
Isao
I
Kobayashi
Katsura
K
Kojima
Keiro
K
Uchino
Hideki
H
Sezutsu
Tetsuya
T
Iizuka
Ken-Ichiro
K
Tatematsu
Naoyuki
N
Yonemura
Hiromitsu
H
Tanaka
Minoru
M
Yamakawa
Eri
E
Ogura
Yusuke
Y
Kamachi
Toshiki
T
Tamura
D000818:Animals; D030801:Animals, Genetically Modified; D012831:Bombyx / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins