title: Heat-inducible transgenic expression in the silkmoth Bombyx mori.
abstract: Germline transformation with new transposon vectors now enables causal tests of gene function via ectopic protein expression or RNA interference in non-drosophilid insects. The problem remains of how to drive the transgene expression in vivo. We employed germline transformation using the piggyBac 3xP3-EGFP vector to test whether the Drosophila heat shock hsp70 promoter will be active in the live silkworm. We modified the original vector by cloning the coding sequence for Bombyx nuclear receptor Ftz-F1 between the hsp70 promoter and the terminator. Three independent transgenic lines expressing the Pax-6-driven EGFP marker in larval and adult photoreceptors were obtained with efficiencies of up to 1.7% of fertile G0 adults that gave GFP-positive progeny. Chromosomal integration of the transposon was confirmed with inverse PCR. Heat induction of the transgenic BmFtz-F1 was proven at both the mRNA and protein levels. RT-PCR data showed that the Drosophila heat shock promoter was functional in all three transgenic lines. Although basal activity was apparent at 25 degrees C, 1 h at 42 degrees C induced BmFtz-F1 mRNA at different stages of development and in diverse tissues. The relative levels of induction differed among the transgenic lines. Northern blot hybridization detected transgenic BmFtz-F1 only after heat shock and low levels of the mRNA were still present 6 h after the heat treatment. Immunostaining of epidermis using anti-BmFtz-F1 antibody showed a clear increase of nuclear signal 90 min after a heat shock.
authors.affiliation: University of South Bohemia, Branisovká 31, Ceské Budejovice 370 05, Czech Republic.
authors.forename: Mirka
authors.initials: M
authors.lastname: Uhlírová
authors.forename: Masako
authors.initials: M
authors.lastname: Asahina
authors.forename: Lynn M
authors.initials: LM
authors.lastname: Riddiford
authors.forename: Marek
authors.initials: M
authors.lastname: Jindra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000235:genetics*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D051717:Fushi Tarazu Transcription Factors; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D018398:Homeodomain Proteins; D006358:Hot Temperature; D019476:Insect Proteins; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D015533:Transcriptional Activation; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018840:HSP70 Heat-Shock Proteins; D018398:Homeodomain Proteins; D019476:Insect Proteins; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; C499713:nuclear hormone receptor FTZ-F1, Bombyx mori