Dev Genes Evol (2002) 212:145–151 DOI 10.1007/s00427-002-0221-8
TECHNICAL NOTE
Mirka Uhlírˇová · Masako Asahina Lynn M. Riddiford · Marek Jindra
Heat-inducible transgenic expression in the silkmoth Bombyx mori
Received: 16 October 2001 / Accepted: 22 January 2002 / Published online: 1 March 2002 © Springer-Verlag 2002
Abstract Germline transformation with new transposon vectors now enables causal tests of gene function via ectopic protein expression or RNA interference in nondrosophilid 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°C, 1 h at 42°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 Edited by D. Tautz M. Uhlírˇová University of South Bohemia, Branišovká 31, Cˇeské Budeˇ jovice 370 05, Czech Republic M. Asahina Institute of Parasitology ASCR, Branišovká 31, Cˇeské Budeˇ jovice 370 05, Czech Republic L.M. Riddiford Department of Zoology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA M. Jindra (✉) Institute of Entomology ASCR, Branišovká 31, Cˇeské Budeˇ jovice 370 05, Czech Republic e-mail:
[email protected] Tel.: +420-38-7775232, Fax: +420-38-5300354
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. Keywords Germline transformation · piggyBac transposon · Heat shock promoter · Nuclear receptor Ftz-F1 · Bombyx mori
Introduction Most functional genetic studies on insect development come from the fruit fly Drosophila melanogaster owing to our ability to manipulate genes with the P-element transposon (Rubin and Spradling 1982). Although Drosophila has many advantages, this species is highly derived and thus may not be representative for studies of some aspects of insect development. Non-drosophilid insects including moths could provide better understanding of processes such as metamorphosis or seasonal rhythms but they have suffered from the lack of genetic methodology. The use of the P-element tools is restricted only to some Drosophila species (O’Brochta and Handler 1988), but recent progress in insect germline transformation with more promiscuous transposon vectors (Handler et al. 1998; Berghammer et al. 1999; Tamura et al. 2000) promises that this drawback is about to be overcome. These vectors now allow genes to be introduced into insect genomes, but the problem arises of how to activate their expression when desired. One good candidate driver for inducible expression is the Drosophila heat shock protein 70 (hsp70) promoter (Thummel and Pirrotta 1992). Lepidopteran larvae have been known to respond to warming by expressing heat shock proteins (Fittinghoff and Riddiford 1990) and studies with two lepidopteran cell lines (Lan and Riddiford 1997; Kimura et al. 1999) encouraged the idea that hsp70 promoter might also be active in vivo. As a marker of heat-induced expression, we chose the nuclear receptor Ftz-F1, a key member of the ecdyste-
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roid signaling pathway that is a focus of our interest (Riddiford et al. 1999). ftz-f1 is required for metamorphosis in Drosophila (Broadus et al. 1999; Yamada et al. 2000) and for molting in both Drosophila and Caenorhabditis elegans (Asahina et al. 2000). Expression of the Bombyx ortholog BmFtz-F1 is restricted by the ecdysteroid rise and decline to short periods during each molt (Sun et al. 1994). Therefore, misexpression at other times may be easily detected. We demonstrate here that piggyBac can deliver DNA inserts large enough to contain both coding and regulatory sequences into the Bombyx genome. Importantly, the expression of a transgene was easily induced in vivo by heat when cloned under the Drosophila heat shock promoter hsp70. For the first time this system allowed temporal control of transgenic activity in a live non-drosophilid insect. It may facilitate studies of genes important for developmental timing, such as BmFtz-F1.
Materials and methods Experimental animals The silkmoth larvae of a non-diapausing strain, Nistari (Tamura et al. 2000), were reared on an artificial diet (Nihon Nosankogyo) at 25°C under a 16 h light: 8 h dark photoperiod.
750 ng/µl) of the pBac{hsFtz-F1} transformation vector and the helper plasmid A3 PIG5-3 (Tamura et al. 2000) in sterile water. Injected eggs were placed into a humidified box at 25°C. Hatched larvae were transferred onto artificial diet and grown to adulthood. G0 adults were mated pairwise with each other or with the parental Nistari strain. G1 embryos were screened for EGFP expression in larval photoreceptors (stemmata) when their heads became pigmented (day 7 at 25°C; Thomas et al. 2002) under an Olympus SZX12 binocular with a fluorescent attachment using the wideband barrier filter with bandpass (Olympus U-MWIBA2, excitation 460–490 nm; transmission range 510–550 nm; splitting wavelength of dichroic mirror 505 nm). Inverse PCR For extraction of genomic DNA, transformed or control (Nistari) second instar larvae were homogenized in a buffer containing 20 mM Tris pH 7.9, 10 mM EDTA pH 8.0, 100 mM NaCl, 0.5% N-lauroylsarcosine and RNase A (25 µg/ml). Proteins were removed by proteinase K treatment and a series of phenol/chloroform extractions. Inverse PCR (iPCR) was performed essentially as described (Hediger et al. 2001) on 3-µg DNA samples digested by HaeIII and circularized with ligase. Primers set RF (5′-TCGATATACAGACCGATAAAACA-3′) and RR (5′-TCAGTCAGAAACAACTTTGGCA-3′) and primers set LF (5′-TGACCTTGCCACAGAGGACTAT-3′) and LR (5′-ACACTTACCGCATTGACAAGCA-3′) were shorter versions of those described in Hediger et al. (2001) and were specific to the 3′and 5′ piggyBac ends, respectively. Amplification was carried out in 35 cycles of 1 min at 94°C, 40 s at 62°C, and 3 min at 72°C. Heat shock treatments
cDNA isolation and sequence analysis of BmFtz-F1 Poly(A)+ RNA was prepared from epidermis of a single molting fourth instar larva, spiracle index E, when BmFtz-F1 mRNA is high (Sun et al. 1994). First strand cDNA was synthesized by SuperScript II reverse transcriptase with oligo(dT) primers (Gibco BRL). The forward 5′-AAgcggccgcTATAATGCACGAAGACGCTC-3′ and reverse 5′-ACAtctagaATCTGTATTGGGACTAGGCA-3′ primers based on the sequence of BmFtz-F1 (Sun et al. 1994) were used for PCR to obtain the entire ORF. PCR products of several independent reactions were cloned into pBluescript II SK vector (Stratagene) and sequenced automatically using the BigDye terminator sequencing kit (Perkin-Elmer). Plasmid construction The heat-inducible BmFtz-F1 cassette was assembled in a pSLfaHSfa vector (a gift from Dr. Ernst Wimmer) prepared from the pSLfa1180fa plasmid (Horn and Wimmer 2000) by inserting the D. melanogaster hsp70 promoter and terminator regions from the pCaSpeR-hs vector (Thummel and Pirrotta 1992). The 1,803-bp fragment of BmFtz-F1 cDNA containing the entire ORF was cloned with NotI and XbaI between the hsp70 promoter and terminator. Then the whole cassette was transferred into pBac{3xP3EGFPafm} vector (Horn and Wimmer 2000) using AscI and FseI restriction enzymes to produce the resulting transformation vector pBac{hsFtz-F1} (Fig. 2). Transformation of B. mori Plasmid DNAs were prepared using the QIAGEN Midi kit and before microinjection were centrifuged through Ultrafree MC 0.1-µm filters (Millipore). The preblastoderm embryos, no older than 2 h after oviposition, were microinjected into the posterior end with a single glass capillary (GDC-1, Narishige) and sealed with acrylic glue. The injection mixture contained equal amounts (500 ng/µl or
Larvae were placed individually either in 1.5-ml microcentrifuge tubes (second instar) or 10-ml plastic tubes (fourth or fifth instar) plugged with cotton such that the larvae could move only in the lower part of the tube. Tubes were then submersed into a water bath so that the cotton was below the water level. Animals were heated for 60 or 90 min at 42°C (Fittinghoff and Riddiford 1990). Following heat shock larvae were returned to food. RNA preparation Total RNA from dorsal abdominal epidermis or from whole larvae was extracted using the TRIZOL Reagent (Gibco BRL). Silk gland RNA was prepared from a single fifth instar posterior gland by homogenization and overnight precipitation in 3 M LiCl, 6 M urea, 10 mM sodium acetate at 4°C. RNA was centrifuged, washed in 4 M LiCl, 6 M urea, and resuspended in RNase-free water. The suspension was extracted with phenol/chloroform and total RNA was precipitated with ethanol. Poly(A)+ RNA from whole larvae was isolated using the Quickprep micro mRNA purification kit (Pharmacia). RNA concentrations were measured spectrophotometrically at 260 nm (A260=1 for 40 µg/ml). Reverse transcriptase PCR For first strand cDNA synthesis, total RNA (1 or 2.5 µg) from dissected tissues or whole animals was reverse-transcribed by SuperScript II with oligo(dT) primers (Gibco BRL) in a reaction volume of 20 µl. From each reaction 2 µl were used as a template for 35 PCR cycles of 94°C (1 min), 60°C (40 s) and 72°C (2 min). Primers HSpro (5′-ACTCTGAATAGGGAATTGGG-3′) and F1rm (5′-TACGGATCCCGCATATGTAATCTGGAACG-3′) were specific for the BmFtz-F1 transgene (Fig. 2). For amplification of control mRNAs, primers were derived from two Bombyx genes, either dopa decarboxylase (AF372836) or mbf1 (AB001078). All samples of total RNA were treated with DNase before RT-PCR to
147 preclude genomic DNA contamination, and no-reverse transcriptase controls were performed for each sample.
the BmCT5 cDNA clone and deposited the corrected version in GenBank (accession number AF426830).
Northern blot hybridization
Transformation experiments
Approximately 150 ng poly(A)+ RNA were separated on a 1% agarose gel containing 2.2 M formaldehyde and transferred onto a positively charged nylon membrane (Roche). Digoxigenin-labeled antisense cRNA probes were synthesized from PCR-amplified cDNA templates with SP6 or T3 RNA polymerases (Roche). The probes were (1) the entire BmFtz-F1 coding sequence, and (2) a Bombyx ribosomal protein RpL3 cDNA (Matsuoka and Fujiwara 2000). High stringency hybridization (68°C, 50% formamide) and washes followed by detection with an anti-DIG alkaline phosphatase and a chemiluminescent substrate were performed according to the DIG northern kit protocol (Roche). Chemiluminescence was visualized by exposure on a Kodak BioMax X-ray film. Immunocytochemistry Whole-mount immunocytochemistry of dorsal epidermis from abdominal segments 5 and 6 was performed as previously described (Asahina et al. 1997), using a polyclonal anti-BmFtz-F1 antibody (a gift from Dr. H. Ueda), diluted 1:200 in PBST containing 1% normal goat serum. A goat anti-rabbit IgG, Cy3-conjugated secondary antibody (1:2,000 in PBST) (Amersham) was used for direct fluorescent detection. Tissues were mounted on slides with DABCO (Fluka) and the preparations were analyzed using a confocal microscope (Zeiss LSM 410) under identical brightness and contrast settings.
We constructed pBac{hsFtz-F1} from the original pBac{3xP3-EGFPafm} transposon vector (Horn and Wimmer 2000) by cloning the BmFtz-F1 coding sequence between the Drosophila hsp70 promoter and the terminator (Fig. 2). As a host for transformation we chose the Nistari strain of the silkmoth, advantageous for its non-diapause character and previous successful transformation (Tamura et al. 2000). From 2,873 eggs coinjected with pBac{hsFtz-F1} and a transposase helper plasmid (see Materials and methods) in three separate trials, we obtained three independent transgenic lines. Transformation efficiency thus varied between 0 and 1.7% of fertile G0 adults that gave EGFP-positive progeny (Table 1). This is low relative to piggyBac transformation efficiencies achieved previously in Nistari silkmoth: 3.9% by Tamura et al. (2000), 17% in Musca domestica (Hediger et al. 2001), 35% in D. melanogaster and even 60% transformants in the flour beetle Tribolium castaneum (Berghammer et al. 1999). The universal activity of the artificial 3xP3-TATA promoter based on Pax-6/Eyeless binding sites (Berghammer et al. 1999) enabled us to detect green fluorescence from mid-embry-
Results and discussion Sequence of B. mori Ftz-F1 Sun et al. (1994) isolated the complete cDNA sequence for the BmFtz-F1 gene. Because we only had a partial BmFtz-F1 cDNA clone, BmCT5 (kindly provided by Dr. H. Ueda), we employed RT-PCR to clone the whole coding sequence. Epidermal mRNA was isolated from larvae during the fourth instar (spiracle index E), when the ecdysteroid titer falls and BmFtz-F1 is induced (Sun et al. 1994). Sequencing of independent RT-PCR clones revealed several conflicts with the originally published BmFtz-F1 cDNA in the 3′ coding region. The resulting amino acid changes lead the BmFtz-F1 C-terminus to a high similarity with the conserved activation domain AF-2 (Fig. 1) found in many types of nuclear receptors (Danielian et al. 1992). This region is vital for receptor interactions with coactivators. The remarkable conservation of AF-2 between Bombyx and mammals suggests that this domain interacts with similar partners in insects and vertebrates. We also confirmed the sequence from
Fig. 1 Comparison of the C-terminal activation domains AF-2 among Ftz-F1 orthologs. Corrected sequence of the Bombyx mori (Bm) Ftz-F1 (AF426830) closely matches the conserved AF-2 (boxed). Ms Manduca sexta (AF288089), Aa Aedes aegypti (AF274870), Dm Drosophila melanogaster (AAF49231), Rn Rattus norvegicus (AB012961), Hs Homo sapiens (AB019246), Ce Caenorhabditis elegans (AF179215) Fig. 2 Design of a piggyBac vector for inducible transgene expression. The marker gene EGFP is located behind the Pax-6 artificial promoter driving expression in photoreceptor cells (Horn and Wimmer 2000. A heat-shock-inducible cassette with BmFtz-F1 transgene was cloned using AscI and FseI restriction enzymes. HSpro and F1rm are primers used for RT-PCR to identify the transgene expression only. For inverse PCR, HaeIII restriction sites and two sets of primers (LF + LR or RF + RR) within the piggyBac arms are shown
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Fig. 3A–D Expression of enhanced GFP marker detected at different developmental stages of silkmoth transformed with the pBac{hsFtz-F1} vector. A Seven-day-old embryo showing green fluorescence in the developing larval stemmata (arrowhead) and nervous ganglia (arrow). Yellow-green color of the rest of the body is caused by autofluorescence. B Adult compound eyes. EGFP
Table 1 Transformation efficiency
a
Both vector and helper plasmids were at the indicated concentrations b Percent of all G0 surviving adults
signal in most of the ommatidia is invisible from one angle due to eye pigmentation. C Arrowhead shows new second instar stemmata in a molting larva. D Stemmata of a newly ecdysed second instar larva (arrowhead). Arrow shows EGFP, cotransported with eye pigment along the axon
Injection round
DNA concentrationa (ng/µl)
No. injected eggs
No. G0 adults
G0 adults giving EGFP+ progeny
Independent transgenic lines
No. EGFP+ G1 larvae
1
500
1,064
116
2 (1.72%)b
2 3
500 750
1,043 766
203 199
0 1 (0.48%)b
Line 26 Line 27 – Line 208
2 6 – 10
ogenesis through the adult stage (Fig. 3) Besides the simple larval eyes (stemmata) and adult compound eyes, the EGFP marker was visible in CNS and ventral ganglia (Fig. 3). All of the larval stemmata and adult ommatidia were EGFP-positive, but the signal in each ommatidium was only visible from a particular angle due to blocking by pigment. Described fluorescence patterns were simi-
lar to those previously seen in flies and silkmoth carrying the same 3xP3 promoter (Horn et al. 2000; Thomas et al. 2002). Transformed lines 26, 27 and 208 all showed a comparable intensity of EGFP fluorescence. Sublines were generated by single matings of each transformant with the parental Nistari strain. The ratio of EGFP-positive
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and negative animals from these backcrosses was 1:1, suggesting that the vector had integrated into a single chromosome. Intercrosses between EGFP-positive siblings gave ratios close to 3:1 (EGFP+:EGFP-) within each line, indicating that pBac{hsFtz-F1} homozygotes were viable. The Mendelian inheritance of EGFP has been stable for at least seven generations in all three transgenic lines. Chromosomal integration of piggyBac has been well documented in several insect species (Handler et al. 1998; Peloquin et al. 2000; Hediger et al. 2001; Kokoza et al. 2001) including B. mori (Tamura et al. 2000; Thomas et al. 2002). We verified the integration directly by sequencing of inverse PCR products from transgenic line 208 with primers LR and RF (Fig. 2). The sequence (gattagcatattaacTTAA-piggyBacTTAAtaagtttatttactt) revealed the characteristic TTAA duplication at the piggyBac junction with chromosomal DNA (Cary et al. 1989). Transgenic expression is rapidly induced in vivo Previous knowledge about the activity of the Drosophila hsp70 promoter outside Drosophila comes merely from transient expression of reporter genes using Manduca (Lan and Riddiford 1997), Bombyx (Kimura et al. 1999) or mosquito (Zhao and Eggleston 1999) cell lines. Although cell lines are extremely useful, they do not fully reflect the regulation of a gene in a live animal. In this study we provide the first evidence that in Bombyx, the Drosophila hsp70 promoter is a good inducible promoter for transgenic approaches in vivo. Individuals of all three transformed lines were tested for heat-inducible expression of transgenic BmFtz-F1 under the Drosophila hsp70 promoter. We treated second or fifth instar feeding or fourth instar molting larvae, transgenic and control, at 42°C for 60 or 90 min, or repeatedly. Samples of whole animals, epidermis or silk glands were collected either immediately after heat shock or 1, 2, 3 or 6 h later. The expression profile of the transgene was analyzed at the mRNA level using RT-PCR. It was critical that the primers detected only the transgenic and not endogenous BmFtz-F1 mRNA. We achieved this by placing the forward primer HSpro into the already transcribed portion of the hsp70 promoter (Fig. 2). If primers derived from the BmFtz-F1 sequence alone, the RT-PCR was saturated with an endogenous mRNA template even in the absence of heat shock. This is consistent with low BmFtz-F1 mRNA levels detected by northern blots during feeding periods (Liu et al. 2000 and Fig. 5). RT-PCR data clearly showed that the Drosophila heat shock promoter was functional in all three transgenic lines upon exposure to 42°C. Figure 4 gives examples of the transgenic mRNA induction at various stages of development and/or tissues, in the epidermis and silk glands; the same results were obtained with all three lines (data not shown). The induction was visible immediately after the heat treatment. Low levels of basal pro-
Fig. 4A–C Heat-shock-induced expression of BmFtz-F1 transgene as detected by RT-PCR. Host strain Nistari (Nist.) and three transgenic lines 26, 27 and 208 were heat shocked at 42°C for 60 min. For Nistari and line 26, day-3 fourth instar molting larvae (spiracle index C) were treated. Larvae of line 27 were treated on day-2 fifth instar and those of line 208 on day-1 second instar, both during the intermolt feeding phase. Total RNA (2.5 µg) from epidermis, silk glands or whole larvae were subjected to RT-PCR with primers HSpro and F1rm (see also Fig. 2), specific only for the transgene (A). The same primers were applied on all RNA samples that had been processed without reverse transcriptase to exclude contamination with genomic DNA (B). Amplification of dopa decarboxylase cDNA served as a positive control for RT-PCR, except for the silk glands where mbf1 gene-specific primers were used (C). n No heat shock; 0, 1, 2, samples taken either immediately, 1 h or 2 h after heat shock; R repeated heat shock (60 min at 42°C plus another 60 min 1 h after the first treatment)
moter activity were apparent at 25°C. Contamination of RT-PCR with genomic DNA was excluded by DNase treatments and no-RT controls for all samples (Fig. 4). We chose line 208 to validate the RT-PCR results and to examine the time course of transgene expression using northern blot hybridization. Figure 5 shows that induction occurred immediately after heat shock, confirming the RT-PCR data. Induced mRNA was detectable for at least 6 h but the level decreased with time. A second heat shock 1 h after the first reinduced high levels (R, Fig. 5). No induction was seen without heat shock. This does not mean that the hsp70 promoter has no activity at 25°C, just that the method is not as sensitive as RT-PCR. On the northern blot, however, the transgenic BmFtz-F1 mRNA can be easily discerned from the endogenous one by size (Fig. 5). To determine whether the transgenic BmFtz-F1 mRNA is also translated into the protein, we stained epidermis of day-1 fifth instar larvae with anti-BmFtz-F1 antibody. Confocal images in Fig. 6 show that nuclear staining in epidermal cells of transgenic animals strongly intensified 90 min after a 90-min heat treatment. Weak signal in the control was possibly caused by a basal activity of hsp70 at 25°C or the presence of endogenous BmFtz-F1 protein (cf. Figs. 4 and 5). Control experiments without the primary antibody showed no staining (data not shown). Heat shock treatment of larvae caused rapid induction of BmFtz-F1 over its low but detectable endogenous
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levels. One might expect to cause some aberrant phenotype by this induction that might help us determine more precisely the role of BmFtz-F1 in molting and metamorphosis. Because some endogenous BmFtz-F1 is present even in the intermolt periods (Fig. 5), it is understandable that no obvious defects resulted from induction of the wild-type version of the protein. However, our heat shock system offers a good possibility of addressing the protein role by inducing its dominant-negative form that is likely to perturb the normal function. Alternatively, conditional gene knock-downs mediated by RNA interference can be achieved via heat induction of transgenic double-stranded RNA as has been done for Drosophila (Lam and Thummel 2000). piggyBac is known to be a versatile vector for insect transformation. We show in this study that the Drosophila hsp70 promoter is functional in vivo in a non-drosophilid species. In summary, the described system provides an important step towards genetic studies of development in a wide variety of insect models.
Fig. 5 Northern blot analysis of heat-induced BmFtz-F1 expression. Control and transgenic (line 208) day-1 second instar larvae were treated at 42°C for 90 min. 150 ng poly(A)+ RNA from whole larvae were hybridized with an antisense BmFtz-F1 cRNA probe. The transgenic mRNA appears at the expected size of around 2.3 kb (arrow). The faint band around 7 kb probably represents the endogenous BmFtz-F1 transcript (arrowhead). The membrane was rehybridized with a control probe for a constitutive ribosomal protein RpL3 (Matsuoka and Fujiwara 2000). NH A transgenic larva without heat shock, asterisk Nistari heat-shocked control, R repeated heat shock (90 min at 42°C plus another 60 min 1 h after the first treatment)
Fig. 6 Immunocytochemistry of epidermis using anti-BmFtz-F1 antibody. Day-1 fifth instar transgenic larvae (line 208) were treated at 42°C for 90 min and dorsal abdominal epidermis was dissected 90 min after the heat shock (right). Epidermis of a transgenic larva without heat shock was used for a control (left). Samples were stained with anti-BmFtz-F1 antibody (dilution 1:200) and Cy3-conjugated secondary antibody (1:2,000). The images were captured by confocal microscopy under identical brightness and contrast settings
Acknowledgements We are grateful to Dr. Ernst Wimmer for providing the pBac{3xP3-EGFPafm} vector and pSLfaHSfa and Dr. Pierre Couble for the A3PIG5-3 transposase helper. We thank Dr. Ivo Sˇauman for excellent technical assistance with confocal microscopy and useful discussions. Kind advice from Dr. JeanLuc Thomas and Dr. Toshiki Tamura on Bombyx transformation and screening is appreciated. The BmFtz-F1 antibody and cDNA clone were generous gifts from Drs. Susumu Hirose and Hitoshi Ueda. The RpL3 cDNA probe was provided by Dr. Haruhiko Fujiwara. This work was supported by a NIH FIRCA grant R03 TWO1209-01 to M.J. and L.M.R., by the Czech Academy of Sciences grant B5007002 to M.J., and from the Institute of Entomology project Z5 007 907 (Acad. Sci. CR).
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