Targeted Gene Expression Using the GAL4/UAS System in the Silkworm Bombyx mori

Corresponding author: Toshiki Tamura, National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba, Ibaraki 305-8634, Japan., ttamura{at}affrc.go.jp (E-mail)

Communicating editor: M. SIMMONS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The silkworm Bombyx mori is one of the most well-studied insects in terms of both genetics and physiology and is recognized as the model lepidopteran insect. To develop an efficient system for analyzing gene function in the silkworm, we investigated the feasibility of using the GAL4/UAS system in conjunction with piggyBac vector-mediated germ-line transformation for targeted gene expression. To drive the GAL4 gene, we used two endogenous promoters that originated from the B. mori actin A3 (BmA3) and fibroin light-chain (FiL) genes and the artificial promoter 3xP3. GFP was used as the reporter. In initial tests of the function of the GAL4/UAS system, we generated transgenic animals that carried the UAS-GFP construct plus either BmA3-GAL4 or 3xP3-GAL4. GFP fluorescence was observed in the tissues of GFP-positive animals, in which both promoters drove GAL4 gene expression. Animals that possessed only the GAL4 gene or UAS-GFP construct did not show GFP fluorescence. In addition, as a further test of the ability of the GAL4/UAS system to drive tissue-specific expression we constructed FiL-GAL4 lines with 3xP3-CFP as the transformation marker. FiL-GAL4 x UAS-GFP crosses showed GFP expression in the posterior silk gland, in which the endogenous FiL gene is normally expressed. These results show that the GAL4/UAS system is applicable to B. mori and emphasize the potential of this system for controlled analyses of B. mori gene function.

TRANSGENIC organisms are powerful tools for the analysis of gene function. The application of transgenic methods to insects was limited to Drosophila melanogaster until recently, mainly because the transposon vector P element, which is used for the transformation of D. melanogaster, has very strong species specificity. Thus, germ-line transformation using the P element has been restricted to species that are closely related to D. melanogaster (HANDLER et al. 1993 ). Recently, several different types of transposons, such as piggyBac, Hermes, Minos, hobo, and mariner, have been identified in insects and have been used successfully as vectors for germ-line transformation in various insect species (HANDLER 2001 ).

The domesticated silkworm (Bombyx mori) is one of a few lepidopteran species that have been used for genetic analysis. Hundreds of different geographical and mutant strains have been preserved in Japan, China, Korea, India, Italy, France, and other countries. Among these strains, >200 mutant genes have been identified. These mutants have been used to construct a linkage map (DOIRA 1992 ) and to analyze gene function (NAGATA et al. 1996 ; KOMOTO 2002 ; MATSUNAGA and FUJIWARA 2002 ; QUAN et al. 2002 ). Moreover, a silkworm genome research program is currently underway. Three bacterial artificial chromosome libraries have been constructed from the silkworm genome (e.g., WU et al. 1999 ), and a silkworm whole-genome sequencing project is about to start. Molecular linkage maps have also been constructed (PROMBOON et al. 1995 ; YASUKOCHI 1998 ; HARA et al. 2001 ; TAN et al. 2001 ; KADONO-OKUDA et al. 2002 ), and these maps will be upgraded as further information becomes available from genomic analyses. The expressed sequence tag (EST) database, which includes >60% of the silkworm genes (K. MITA, personal communication), is currently available (SilkBase: http://www.ab.a.u-tokyo.ac.jp/silkbase/), and cDNA microarrays have been produced from 6000 ESTs (K. MITA, personal communication). Moreover, as an experimental animal the silkworm has the advantages that it is easily handled, the larvae are highly adapted for artificial rearing, and the adult moths are unable to fly. Thus, the silkworm is regarded as a model insect for the Lepidoptera in particular. However, since transformation of silkworms was not achieved until recently, its utility for gene functional analyses was limited.

In 2000, we developed a germ-line transformation method for the silkworm using the transposable element piggyBac as the vector (TAMURA et al. 2000 ). To date, we have successfully introduced several genes into silkworms, and we have used these transformants to analyze gene function and to elucidate physiological phenomena (S. INOUE and M. IMAMURA, unpublished data). We now wish to extend our studies in the silkworm to the adaptation of the GAL4/upstream activating sequence (UAS) system (FISCHER et al. 1988 ; BRAND and PERRIMON 1993 ), which is a powerful technique for unraveling gene function. The GAL4/UAS system has been used routinely in Drosophila (BRAND and PERRIMON 1993 ) and has also been adapted to the mouse (ORNITZ et al. 1991 ), zebrafish (SCHEER and CAMPOS-ORTEGA 1999 ), Xenopus (HARTLEY et al. 2002 ), and Arabidopsis (GUYER et al. 1998 ). This technique relies on the generation of transgenic lines that carry an activator or effector construct. The activator lines express the GAL4 yeast transcription factor under the control of a test promoter, whereas the effector lines contain the GAL4-binding sequence linked to the gene of interest (BRAND and PERRIMON 1993 ).

The GAL4/UAS system has certain advantages. First, it enables one to analyze simultaneously the effects of a single transgene selectively in different tissues and at different developmental stages. Conversely, it can also be used to study several different genes in a particular tissue or cell or at a specific time point. Second, this system makes possible the generation of transgenic lines that carry lethal genes or genes for toxic proteins and enables the functional analysis of these genes as well as the targeted destruction of a cell or tissue. Third, the GAL4 system can be used to amplify the expression level of a transgene.

In this study, we demonstrate the feasibility of using the GAL4/UAS system in combination with the piggyBac transposon vector in the silkworm, by showing that the green fluorescent protein (GFP) gene is expressed in a predictable tissue-specific pattern in the progeny of crosses between the GAL4 and UAS-GFP lines. This study emphasizes that the GAL4 system using the piggyBac vector is also applicable to non-drosophilid insects that have undergone successful germ-line transformation with the piggyBac vector.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Silkworm strains:
The w1-pnd strain, which is nondiapausing and has nonpigmented eggs and eyes, was used in these experiments. The eggs of this strain develop to the larval stage, without termination of development, 11 days after the injection of DNA. The larvae were reared on an artificial diet (Nihon Nosanko) at 25°. This strain is maintained at the National Institute of Agrobiological Sciences.

Construction of vectors:
The plasmids (Fig 1) were constructed as described below.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Organization of the GAL4 and UAS constructs derived from the piggyBac transposon element. The GAL4 promoter fragments were inserted into the HpaI site of p3E1.2. The 3xP3-CFP fragment was inserted into the BglII site to produce pBacFiL-GAL4/3xP3-CFP. pBacUAS-GFP was constructed from pPIGA3GFP.

pBacUAS-GFP: pBacUAS-GFP was constructed from pPIGA3DsRed1b, which was designed to identify organs and cells in the transplantation experiment. The BamHI-NotI fragment of pPIGA3GFP (TAMURA et al. 2000 ), which contains the EGFP sequence, was replaced with the BamHI-NotI fragment from pDsRed1-N1 (CLONTECH, Palo Alto, CA), which contains the DsRed1 sequence, to yield the plasmid pPIGA3DsRed1a. To delete the polylinker sequence 5'-GAATTCGAGCTCGGTACCCGGGGATCCTCTAGA-3', which contains EcoRI, SacI, KpnI, SmaI, BamHI, and XbaI restriction sites, from vector pPIGA3DsRed1a, PCR was conducted with pfu DNA polymerase (Stratagene, La Jolla, CA) using pPIGA3DsRed1a plasmid DNA as the template. The nucleotide sequences of the primers were 5'-GGCGTCGACGTAATCATGGTCATAGCTGTTTCC-3' (forward primer) and 5'-GCACGCGTTCGTGTACAGACGTA-3' (reverse primer). The PCR conditions were initial denaturation at 94° for 2 min, 30 cycles of 94° for 30 sec, 55° for 30 sec, and 72° for 3 min, followed by 72° for 5 min. The amplified fragment was digested with SalI and MluI and then ligated with the 4.9-kb SalI-MluI fragment derived from pPIGA3DsRed1a, which contained the BmA3 promoter fragment and the DsRed1 gene. The resulting plasmid was named pPIGA3DsRed1b. The HindIII-EcoRI fragment containing the UAS (the GAL4-upstream activating sequence) and TATA element of the D. melanogaster heat-shock protein 70 (Dmhsp70) promoter (BRAND and PERRIMON 1993 ) was subcloned into pEGFP-N1 (CLONTECH). The UAS-EGFP fragment was excised using XhoI and NotI and inserted into the NotI-XhoI site of pPIGA3DsRed1b to yield plasmid pBacUAS-GFP.

pBacBmA3-GAL4: The 647-bp fragment that lies upstream of the ATG start codon of the B. mori cytoplasmic actin A3 gene (BmA3) was amplified by PCR and used as the BmA3 promoter. The nucleotide sequences of the primers were as follows: 5'-GGCGCGCCTCGAGCTCAAGCTTGATG-3' (forward primer) and 5'-GGATCCCTTGAATTAGTCTGCAAG-3' (reverse primer). The recognition sequences for AscI and BamHI were added to the forward and the reverse primer, respectively. PCR was conducted with LA Taq (Takara) using the pPIGA3GFP plasmid DNA as a template. The PCR cycling conditions were as follows: initial denaturation at 95° for 2 min, 30 cycles of 95° for 30 sec, 55° for 30 sec, and 72° for 40 sec, followed by 72° for 7 min. The amplified fragment was subcloned in the pGEM-T Easy vector (Promega, Madison,WI), and the constructs were digested with BamHI and SacII. The BamHI-SacII fragment, which contained the GAL4 gene and Dmhsp70 terminator that originated from pGaTB (BRAND and PERRIMON 1993 ), was inserted into the BamHI-SacII site of the pGEM-T Easy vector containing the BmA3 promoter fragment. The fragment that contained the BmA3-GAL4 gene was excised from this plasmid by digestion with NotI and blunt-end ligated into the HpaI site of p3E1.2 that was the plasmid containing an intact piggyBac transposon element (CARY et al. 1989 ; FRASER et al. 1995 ).

pBac3xP3-GAL4: The 251-bp fragment that included the 3xP3 promoter was obtained by PCR using pBac[3xP3-EGFPafm] (HORN and WIMMER 2000 ) as the template. The nucleotide sequences of the primers were as follows: 5'-AATATGCGAATTCGAGCTCGCCCGGGGATCTAATTC-3' (forward primer) and 5'-TGCAGGAATTCGGGCCCGCGGTACCGTCGACTCTAGC-3' (reverse primer). Single EcoRI sites were added to both primers. PCR was carried out as follows: initial denaturation at 95° for 2 min, 30 cycles of 95° for 30 sec, 55° for 30 sec, and 72° for 30 sec, followed by 72° for 7 min. The 3xP3 promoter fragment was subcloned into the pGEM-T Easy vector. The BamHI and SacII fragment that contained GAL4 and the Dmhsp70 terminator, which was excised from pGaTB, was inserted into the BamHI and SacII sites of pBluescript II SK- (Stratagene). The 3xP3 promoter fragment was excised with EcoRI from the TA vector and inserted into the EcoRI site of the pBluescript II SK- derivative that already contained the GAL4 gene and Dmhsp70 terminator. The 3xP3-GAL4 fusion was removed as a NotI-EcoRV fragment from this plasmid and blunt-end ligated into the HpaI site of p3E1.2.

pBacFiL-GAL4/3xP3-CFP: To amplify the 740-bp region upstream of the fibroin light chain (FiL) gene, PCR was conducted using the plasmid that contained the FiL gene (KIKUCHI et al. 1992 ) as the template, using the following primers: 5'-GGCGCGCCTGCATATTGGACATCC-3' (forward primer) and 5'-CGCGGATCCTTTAGTGGTCTGTTA-3' (reverse primer). The AscI and BamHI sites were attached to the forward and the reverse primers, respectively. The PCR cycling conditions were as follows: initial denaturation at 95° for 2 min, 30 cycles of 95° for 30 sec, 55° for 30 sec, and 72° for 40 sec, followed by 72° for 7 min. The amplified fragment was subcloned into pGEM-T Easy, and the BamHI-SacII fragment of the GAL4 gene from pGaTB was inserted into the BamHI and SacII site of this plasmid. The fragment that contained the FiL-GAL4 gene was excised by digestion with NotI and blunt-end ligated into the HpaI site of p3E1.2 from which superfluous EcoRI, SacI, KpnI, SmaI, and BamHI sites were removed. The resultant plasmid was named pBacFiL-GAL4. Then, to introduce a transformation marker into pBacFiL-GAL4, the 3xP3-ECFP-SV40 terminator fragment was amplified by PCR using pBac[3xP3-ECFPafm] plasmid DNA as the template and the following primers: 5'-CAAGATCTAATTCGAGCTCGCCCGGGGATCTAATTC-3' (forward primer) and 5'-TAGCAGATCTGTACGCGTATCGATAAGCTTTAAG-3' (reverse primer). Both primers had BglII sites at their 5'-ends. PCR was performed as follows: initial denaturation at 95° for 2 min, 30 cycles of 95° for 30 sec, 55° for 30 sec, and 72° for 30 sec, followed by 72° for 7 min. The PCR product was digested with BglII and cloned into the BglII site of pBacFiL-GAL4.

All the PCR products and constructed plasmids were verified by sequencing using an ABI310 or ABI377 DNA sequencer and the BigDye termination DNA sequencing kit (PE Applied Biosystems, Foster City, CA).

Injection of DNA into embryos and detection of GFP and CFP fluorescence:
Plasmid DNA for injection was purified using a plasmid purification kit (QIAGEN, Valencia, CA). pHA3PIG (TAMURA et al. 2000 ) was used as the helper plasmid for the production of transposase. Vector and helper plasmids (each 0.2 µg/µl) were resuspended in 0.5 mM phosphate buffer (pH 7.0), 5 mM KCl, and injected into eggs that were collected between 3 and 5 hr after egg oviposition. In the transient assay, only the vector plasmids were injected. GFP and CFP (the spectral variant of GFP, cyan fluorescent protein) fluorescence was observed under a fluorescence microscope that was equipped with filter sets for GFP2 and CFP (Leica), respectively. Transient expression of the injected DNA was observed in the G₀ eggs 3 days after injection. Screening was performed at a late stage of embryonic development for transformants that were driven by the 3xP3 promoter and in the first instar larvae for transformants that carried the BmA3 promoter.

Preparation of genomic DNA and Southern blot analysis:
Genomic DNA was extracted from adult moths by the SDS-phenol method (OHSHIMA and SUZUKI 1977 ). The DNA (4 µg) was digested with restriction enzymes and fractionated on an 0.8% agarose gel. XhoI and KpnI were used to digest the genomic DNA of the BmA3-GAL4, 3xP3-GAL4, and UAS-GFP strains, and BglII was used to digest the genomic DNA of the FiL-GAL4 strain. The DNA samples were transferred to a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) and fixed by UV cross-linking. Hybridization was performed using the Alkphos direct labeling and detection system (Amersham Pharmacia Biotech). The probes for the GAL4 and GFP genes were prepared from the 2500-bp ClaI fragment of pGaTB and the 1200-bp XhoI-NotI fragment of pBac UAS-GFP, respectively.

PCR detection of transgenes:
To distinguish larvae with single GAL4 or GFP genes from the GFP-negative G₂ larvae, PCR was carried out using 50 ng of genomic DNA from the hemocytes of a single larva as the template. Genomic DNA was prepared using the DNeasy tissue kit (QIAGEN). The following primers were used for gene detection: for the GFP gene, 5'-CTCGTCCTTCAGTGATAGCAG-3' (forward) and 5'-CGCTTAACATGATGGAGCATCG-3' (reverse) and for the GAL4 gene, 5'-CACATGAAGCAGCACGACTTCTTC-3'(forward) and 5'-CTTGATGCCGTTCTTCTGCTTGTC-3' (reverse). PCR was carried out as follows: initial denaturation at 95° for 2 min, 30 cycles of 95° for 30 sec, 63° for 30 sec, and 72° for 30 sec, followed by 72° for 7 min.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Trans-activation of the UAS-GFP gene by the GAL4 promoter element in silkworm embryos in a transient expression assay:
To investigate whether the GAL4/UAS system worked in the silkworm, we first performed a transient expression assay in the embryos. To date, three promoters have been reported to work in transgenic silkworms: the B. mori cytoplasmic actin promoter (BmA3; TAMURA et al. 2000 ), the artificial 3xP3 promoter (THOMAS et al. 2002 ), and the promoter of the D. melanogaster heat-shock protein 70 gene (Dmhsp70; UHLIROVA et al. 2002 ). The BmA3 promoter has been used to drive gene expression in many types of cells at all developmental stages, and the 3xP3 promoter has been shown to stimulate the expression of introduced genes in the cells of stemmata and compound eyes, as well as certain cells of the CNS (HORN et al. 2000 ). We constructed two GAL4 driver plasmids, which were under the control of the BmA3 and 3xP3 promoters (pBacBmA3-GAL4 and pBac3xP3-GAL4), and a UAS reporter plasmid that contained the UAS-GFP fusion gene (pBacUAS-GFP; Fig 1). High levels of GFP expression were observed following the injection of either pBacBmA3-GAL4 or pBac3xP3-GAL4 with pBacUAS-GFP into the embryos (Fig 2). GFP fluorescence was not generated when the plasmids were injected independently. These results showed that transactivation of the UAS-GFP gene by GAL4 occurred during transient expression in silkworm embryos.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Transient expression of the GFP gene in embryos using the GAL4 and UAS constructs. (Left) GFP-fluorescent image of eggs that were injected with DNA constructs; (right) corresponding bright-field image. The plasmids (each 200 µg/µl) were injected into 3- to 4-hr-old embryos, and the embryos were observed 3 days after injection.

The GFP expression of eggs that were co-injected with pBacBmA3-GAL4 and pBacUAS-GFP was much stronger than that of eggs that were injected with pPIGA3GFP that have the GFP gene under direct control of the BmA3 promoter. Similarly, eggs that were co-injected with pBac3xP3-GAL4 and pBacUAS-GFP also showed high levels of GFP expression, although the GFP fluorescence was poor when a single 3xP3 promoter construct, pBac[3xP3-EGFPafm], was injected into the silkworm embryos (data not shown). These results suggest that the regulation of expression by the BmA3 and 3xP3 promoters is enhanced in the GAL4/UAS system.

BmA3-GAL4 and 3xP3-GAL4 both drive the expression of the UAS-GFP gene in transgenic silkworms:
Next, we carried out experiments to show that the GAL4/UAS system functioned in transgenic silkworms (Fig 3). When we started this study, only two promoters (BmA3 and 3xP3) and one fluorescent marker (GFP) had been reported to function in transgenic silkworms. Therefore, we developed the following strategy to show that the GAL4/UAS system applies to the silkworm. First, we established transformants that carried both the promoter-GAL4 and UAS-GFP genes with no marker gene for transformation. If these transformants produce GFP fluorescence, then the GAL4/UAS system functions in the silkworm. However, it is also necessary to prove that transactivation by GAL4 occurs when GAL4 and the UAS-GFP gene coexist as a result of mating. Therefore, crossing experiments were done to recover transformants with only the GAL4 or UAS-GFP gene. GFP-positive G₁ animals were backcrossed to the w1-pnd strain, to generate GFP-negative G₂ animals with only the GAL4 or UAS-GFP gene. Then, the GAL4 and UAS lines were crossed, because if GFP-positive G₃ animals emerged in the ratio of one to three this would prove that the GAL4/UAS system applies to the silkworm transgenic system.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 3. System for testing the GAL4/UAS system in B. mori. First, the promoter-GAL4 and UAS-GFP plasmid vectors were co-injected into embryos so that no transformation marker was needed. If the GAL4/UAS system functions properly in the transgenic silkworms, then GFP-positive animals should be recovered in G1. Next, a crossing experiment was performed to confirm that the GAL4/UAS system can function when GAL4 and UAS coexist as a result of crossing.

The pBacBmA3-GAL4 construct was injected, along with pBacUAS-GFP and the pHA3PIG helper plasmid as a source of transposase, into 1500 eggs of the w1-pnd strain. About 270 G₀ fertile adults were recovered, and they were sibling mated to decrease the number of broods for screening. As a result of screening of 112 broods, 3 broods with GFP-positive larvae were identified (2.7%; Table 1). Similarly 1800 eggs were injected with pBac3xP3-GAL4. After sibling mating of 270 G₀ adults, 121 broods were obtained, and 3 broods with GFP-positive larvae were identified (2.5%). In any GFP-positive individuals, GFP fluorescence was observed in tissues in which both promoters were expected to drive GAL4 gene expression (Fig 4). The frequency of G₁ GFP-positive larvae in the broods from G₀ moths that were injected with the two plasmids, pBacBmA3-GAL4 and pBacUAS-GFP, was between 0.4 and 2.1%; it was between 0.4 and 17.7% for broods from G₀ moths that were injected with the pBac3xP3-GAL4 and pBacUAS-GFP. Unfortunately, the G₁ GFP-positive animals in brood 3 produced by mating moths injected with pBacBmA3-GAL4 and pBacUAS-GFP and brood 1 produced by mating moths injected with pBac3xP3-GAL4 plus pBacUAS-GFP were lost before they became moths.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Transgenic silkworms expressing the GFP gene under the control of the GAL4/UAS system. (A) Fluorescent images of 3-day-old first instar larvae that carry the BmA3-GAL4 and UAS-GFP genes (right) and host-strain w1-pnd larvae as controls (left). (B) Fluorescent and (C) bright-field images of an adult that carries the 3xP3-GAL4 and UAS-GFP genes. (D) Fluorescent and (E) bright-field images of an adult w1-pnd moth as the control.

Southern blot analysis was performed on the genomic DNAs of transformed G₁ animals to identify differences in the insert positions and copy numbers of the transgenes. Five fertile GFP-positive adults in broods 1 and 2, whose parents were injected with pBacBmA3-GAL4 and pBacUAS-GFP, were found to carry single copies of the GAL4 and UAS-GFP genes (Fig 5). The banding patterns were identical for all the transformants (data not shown), which indicated that all of the transformants that carried the BmA3-GAL4 and UAS-GFP genes were produced from the same parent. The finding that two different broods possess the same insertion can be explained by the fact that the G₀ males were repeatedly mated with females because of the limited number. The line that contained the BmA3-GAL4 and UAS-GFP genes is referred to as the A3 line. Twenty-seven and 41 G₁ fertile adults with pBac3xP3-GAL4 and pBacUAS-GFP were recovered from GFP-positive broods 2 and 3, respectively. Southern blot analysis was carried out on the genomic DNA samples of 24 adults from each brood (data not shown). In brood 2, we found two types of transformant with single GAL4 and UAS-GFP genes inserted at different positions, which we refer to as the P2-1 and P2-2 lines, respectively. On the other hand, there were three patterns of integration in brood 3. Although all of the transformants from brood 3 carried an identical single insertion of the GAL4 gene, the UAS-GFP gene appeared in three different patterns: two patterns had single insertions at independent sites and the remaining pattern contained both inserts (Fig 5). We designate these lines as the P3-1, P3-2, and P3-3 lines, respectively.

View larger version (69K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Southern blot analysis of transgene integration patterns in G1 GFP-positive silkworms. Genomic DNA samples from G1 GFP-positive and w1-pnd adults were digested with XhoI and KpnI, separated by agarose gel electrophoresis, and hybridized with GAL4- and GFP-specific probes. The individual DNA hybridization patterns of the w1-pnd, A3, P2-1, P2-2, P3-1, P3-2, and P3-3 lines are shown. Asterisks and solid circles denote the signals for the GAL4 and GFP probes, respectively.

To recover animals that contained only the promoter-GAL4 gene or only the UAS-GFP gene, we backcrossed the G₁ transformants with the w1-pnd host strain. The ratio of the GFP-positive and negative G₂ first instar larvae in all crosses was 1:3 (Table 2). Twenty-four GFP-negative fifth instar larvae were chosen randomly from each line, genomic DNA was prepared from the hemocytes of these animals, and PCR was performed using the GAL4 and GFP gene-specific primers to check their genotypes (Fig 6). Thus, we obtained individuals with either a single GAL4 or UAS-GFP gene. Although the segregation ratios of the genotype varied widely in 24 investigated GFP-negative larvae, they were shown to fit a 1:1:1 ratio by chi-square statistical analysis (Table 2). This result suggested that the GAL4 and UAS-GFP genes were dispersed throughout the transgenic chromosomes.

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Figure 6. PCR screening of G2 transformants that carried single GAL4 or UAS-GFP genes. Genomic PCR was conducted to identify individuals that carried a single GAL4 or UAS-GFP gene. Part of the screening process is shown. Genomic DNA was prepared from hemocytes of GFP-negative fifth instar larvae that were derived from a backcross of GFP-positive G1 with w1-pnd. PCR was carried out using the GAL4-, GFP-, and actin A3-specific primers with genomic DNA as the template. Each lane shows the PCR product from a single larva.

Moths from the four GAL4 lines (one with the BmA3-GAL4 gene from the A3 line and three with the 3xP3-GAL4 gene from the P2-1, P2-2, and P3-1 lines, respectively) were crossed with the UAS-GFP line that carried a single UAS-GFP gene from the A3 line. In the offspring (G₃), 25% of the larvae had acquired GFP-dependent fluorescence, whereas both parents were GFP negative, and the segregation ratio of the genotypes was 1:1:1:1 (Table 3). Southern blot analysis of genomic DNA samples of the G₃ GFP-positive individuals showed that all of them carried both the GAL4 gene and the UAS-GFP gene from the G₂ lines (Fig 7). These results demonstrate that the GAL4/UAS system functions in the silkworm, even when the GAL4 and UAS-GFP genes coexist after crossing.

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Analysis of G3 GFP-positive animals. (A) Southern blot analysis of G3 GFP-positive animals and of G2 individuals that carry a GAL4 or UAS-GFP gene. A3, P2-1, P2-2, and P3-1 indicate the origin of the GAL4 gene borne by each individual. Asterisks and solid circles denote the DNA fragments that hybridized with the GAL4 and GFP probes, respectively. Genomic DNA was double digested with XhoI and KpnI. (B) Fluorescent images of the G3 GFP-positive animals. In A3, the three upper larvae are transformants that carry both the BmA3-GAL4 and the UAS-GFP genes, and the lower larva is a w1-pnd individual. P2-1, P2-2, and P3-1 show moths that carry the UAS-GFP gene plus the 3xP3-GAL4 gene that originated in the P2-1, P2-2, or P3-1 lines, respectively.

Evaluation of the GAL4/UAS system in the transgenic silkworm using the fibroin L-chain promoter:
We generated a GAL4 line that carried GAL4 gene driven by a promoter derived from the FiL gene and the 3xP3-CFP gene as a fluorescent transformation marker. We then investigated the utility of the 3xP3-CFP marker and whether the FiL promoter specifically drives gene expression via the GAL4/UAS system in the posterior division of the silk gland (PSG). The pBacFiL-GAL4/3xP3-CFP construct (Fig 1) was injected with helper plasmid DNA into 1006 eggs, and 19 broods with CFP-positive individuals were obtained (Table 4A; Fig 9A and Fig B). Adult moths from three different CFP-positive broods were backcrossed with the w1-pnd strain and established as the FiL1, FiL2, and FiL3 lines. Southern analysis of the G₂ progeny showed that the FiL1 and FiL2 lines each had a single copy of the GAL4 gene and that the FiL3 line contained two copies of the gene (Fig 8). We found two copies of the GAL4 gene in 12 individuals of the FiL3 line (data not shown), suggesting they were tightly linked. Although the transgenic first instar larvae had five CFP-fluorescent stemmata (Fig 9C and Fig D), GFP fluorescence was not detected (Fig 9E and Fig F; middle). We then crossed these GAL4 lines with the UAS-GFP line, which was heterozygous for the transgene (Fig 8). The ratio of the CFP-positive, CFP/GFP-positive, and negative larvae in all crosses was 1:1:2 (Table 4B). This result supports the notion that the GAL4 genes in the FiL3 line were tightly linked. In the progeny of these crosses, 25% of the larvae gave strong GFP fluorescence on the side where the silk glands were located (Table 4B; Fig 9E and Fig F). Subsequently, the silk glands were dissected from 5-day-old fifth instar larvae and observed with a fluorescent microscope. Very strong GFP fluorescence was detected in the PSG of all the GFP-positive individuals (Fig 9, G–J), but not in GFP-negative individuals (Fig 9K and Fig L). Interestingly, the PSG in the FiL1, -2, and -3 lines was shortened and appeared knotted (Fig 9G and Fig H), while the PSG in the other line was normal (Fig 9I and Fig J). This abnormality in PSG was thought to be caused by cell deformation.

View larger version (126K): [in this window] [in a new window] [Download PPT slide]   Figure 8. Southern blot analysis of FiL-GAL4 lines. Genomic DNA was digested with BglII. FiL1, FiL2, and FiL3 in G2 indicate the individuals that were used in crosses with the UAS-GFP line, and FiL1, FiL2, and FiL3 in G3 represent the GFP-positive individuals that resulted from these crosses, respectively. Asterisks and solid circles denote the DNA fragments that hybridized with the GAL4 and GFP probes, respectively. The arrow shows the 3xP3-CFP fragment that was excised from the GAL4 constructs by digestion with BglII. The signal for the 3xP3-CFP fragment is stronger in the FiL3 line than in the two other lines because this line has two copies of the GAL4 gene.

View larger version (53K): [in this window] [in a new window] [Download PPT slide]   Figure 9. Fluorescent images of transgenic silkworms that carry the FiL-GAL4 gene. (A and B) Seven-day-old embryos of transformants that carry the FiL-GAL4 gene with 3xP3-CFP as a marker and nontransformants (A, CFP-fluorescent image; B, bright-field image). Arrows indicate the developing larval stemmata of the transformants. (C and D) The heads of 2-day-old first instar larvae that carry both the FiL-GAL4/3xP3-CFP and UAS-GFP genes (GAL4/GFP); only the FiL-GAL4/3xP3-CFP gene (GFP) and w1-pnd (Cont.) are shown (C, CFP-fluorescent image; D, bright-field image). (E and F) Two-day-old first instar larvae (E, GFP-fluorescent image; F, bright-field image). Top, transformant that carries both the FiL-GAL4/3xP3-CFP and UAS-GFP genes; middle, transformant that carries only the FiL-GAL4/3xP3-CFP gene; bottom, nontransformant. (G–L) Silk glands of transgenic fifth instar larvae that carry the FiL-GAL4/3xP3-CFP and UAS-GFP genes (G–J) and a w1-pnd larva (K and L; G, I, and K, GFP-fluorescent images; H, J, and L, bright-field images). G and H are the silk gland from an abnormal SG line (FiL1 line), and I and J are the silk gland from a normal SG line. High-level GFP expression is observed only in the PSG of the transformants, which appear green even under bright-field microscopy.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this study, we successfully constructed the GAL4/UAS system in the transgenic silkworm and showed that it could be used to express the GFP gene. The transgenes were normally inherited in a Mendelian manner in all of the GAL4 lines (BmA3-GAL4, 3xP3-GAL4, FiL-GAL4) and in the UAS-GFP line, which indicated that the viability of these lines was not affected by the expression of the transgene. These findings demonstrate that the GAL4/UAS system can be used for targeted transgene expression in silkworms. To date, the GAL4/UAS system has been shown to function in D. melanogaster (FISCHER et al. 1988 ; BRAND and PERRIMON 1993 ), mice (ORNITZ et al. 1991 ), Arabidopsis (GUYER et al. 1998 ), zebrafish (SCHEER and CAMPOS-ORTEGA 1999 ; KOSTER and FRASER 2001 ), and frogs (HARTLEY et al. 2002 ). Our study represents the first attempt to show that the GAL4/UAS system is applicable to non-drosophilid insects. Recently, piggyBac-mediated germ-line transformation has been used successfully in various insects, such as the medfly, Ceratitis capitata (HANDLER et al. 1998 ); the red flour beetle, Tribolium castaneum (BERGHAMMER et al. 1999 ); the pink bollworm, Pectinophora gossypiella (PELOQUIN et al. 2000 ); the Oriental fruit fly, Bactrocera dorsalis (HANDLER and MCCOMBS 2000 ); the Caribbean fruit fly, Anastrepha suspensa (HANDLER and HARRELL 2001 ); the housefly, Musca domestica (HEDIGER et al. 2001 ); the yellow fever mosquito, Aedes aegypti (KOKOZA et al. 2001 ); the malaria mosquito, Anopheles stephensi (NOLAN et al. 2002 ); and the Australian sheep blowfly, Lucilia cuprina (HEINRICH et al. 2002 ). Therefore, the GAL4/UAS system with the piggyBac vector should also be applicable to these insects.

In the transgenic silkworm B. mori, the actin A3 (BmA3) and artificial 3xP3 promoters had been used to drive the expression of the GFP gene (TAMURA et al. 2000 ; THOMAS et al. 2002 ). In this study, we initially used the BmA3 and the 3xP3 promoter to drive the GAL4 gene and showed that these promoters produced sufficient amounts of GFP for imaging purposes. However, the GFP expression level in the transgenic silkworm did not reflect precisely the levels of amplified expression in transient assays of the embryos (Fig 2). It has been reported that transgene expression by GAL4 is somewhat weaker than expected in transgenic zebrafish (SCHEER and CAMPOS-ORTEGA 1999 ) and transgenic Xenopus (HARTLEY et al. 2002 ). To increase transgene expression via the GAL4/UAS system in silkworms, the following modifications may be useful: (1) the introduction of an insulator to both the GAL4 and UAS constructs (BAROLO et al. 2000 ); (2) the insertion of several copies of the UAS-linked gene of interest into the chromosomes (KOSTER and FRASER 2001 ); and (3) the use of the GAL4-VP16 protein, which is a fusion of the GAL4 DNA-binding domain and the herpes simplex virus transcriptional-activation domain VP16 (SADOWSKI et al. 1988 ).

The transformation efficiency of the FiL-GAL4 line was 11% (as a percentage of all the G₀ broods). This value is much higher than that reported previously for transgenic silkworms (TAMURA et al. 2000 ; THOMAS et al. 2002 ; UHLIROVA et al. 2002 ). This is probably due to an improved injection method (T. TAMURA, G. X. QUAN, T. KANDA and N. KUWABARA, unpublished data). Recently, it has been reported that the mobilization frequency of the Minos transposable vector using in vitro synthesized mRNA as the source of transposase is 10-fold higher than that obtained using a helper plasmid (KAPETANAKI et al. 2002 ). This suggests that the application of in vitro synthesized piggyBac transposase mRNA may further increase the efficacy of piggyBac-mediated transformation of silkworms.

Although it has been reported that CFP fluorescence driven by the 3xP3 promoter in D. melanogaster is weaker than the fluorescence of GFP and YFP (the spectral variant of GFP, yellow fluorescent protein; HORN and WIMMER 2000 ), we used this as a marker in the generation of FiL-GAL4-transgenic silkworm lines. Indeed, the CFP-fluorescence intensity of the FiL-GAL4 lines was weaker than that of GFP fluorescence in animals that were transformed with pBac[3xP3-GFPafm] (data not shown). However, screening with the 3xP3-CFP gene is possible during late embryonic stages when the signal is weakest at all the stages, since both the eyes and the eggs of the w1-pnd strain are nonpigmented. Therefore, the 3xP3-CFP gene can be used as a transformation marker, at least in this strain. However, when the stemmata and compound eyes of CFP-positive animals were observed with the GFP2 longpass filter set (Leica), it was difficult to distinguish CFP-positive animals from transgenic animals that carried the 3xP3-GFP gene (data not shown). Thus, care is needed in identifying the tissues and organs that express the reporter gene when CFP and GFP are used as marker and reporter, respectively, in the same individual. CFP and DsRed2 or CFP and YFP are considered to be good marker combinations because these have well-separated excitation and emission spectra (HORN et al. 2002 ).

Abnormal PSGs that expressed GFP were observed in larvae of the three FiL-GAL4 lines that were used in some experiments (FiL1, -2, and -3 lines; Fig 9G and Fig H). This abnormality was also observed in larvae that carried only the FiL-GAL4 genes (data not shown), which suggests that it was caused by GAL4 production. Furthermore, these lines formed no cocoon or a very thin-layer cocoon that resembled those formed in the fibroin-secretion-deficient mutants Nd-s^D and Nd-s (TAKEI et al. 1987 ; MORI et al. 1995 ; data not shown). On the other hand, one FiL-GAL4 line formed normal PSG and a cocoon that was only slightly thinner than that of the wild type. The difference between normal and abnormal FiL-GAL4 lines may be due to differences in the expression levels of the GAL4 gene, as evidenced from the comparison of the relative GFP intensities (Fig 9, G–J). Approximately 200–300 cells in the PSG are specialized for mass production and secretion of the fibroin H- and L-chain proteins together with fibrohexamerin (P25; AKAI 1976 ; TANAKA et al. 1993 ; INOUE et al. 2000 ). Although it is known that GAL4 can be expressed in many cells and tissues of transgenic animals without any toxic effects, it is possible that the mass production of GAL4 in a tissue that is so highly specialized for protein production can be especially disruptive.

Various gene analysis systems using the GAL4/UAS system have been developed in Drosophila. These include enhancer trapping (BRAND and PERRIMON 1993 ), gain-of-function mutagenesis (RORTH 1996 ; TOBA et al. 1999 ), and gene silencing by interference using hairpin-loop RNA (KENNERDELL and CARTHEW 2000 ). In this study, we showed that the GAL4/UAS system is applicable to the silkworm. Recently it was shown that insertional mutagenesis and enhancer trapping is possible using the piggyBac vector in D. melanogaster (HORN et al. 2003 ). Therefore, in the near future we will be able to develop novel systems that employ the GAL4/UAS system for gene discovery and gene functional analysis in the silkworm. Once such systems are constructed, they will contribute to the advance of functional genomics for the silkworm and to comparative and functional genomics for lepidopteran species.

	FOOTNOTES

¹ Present address: National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
² Present address: RIKEN Brain Science Institute, 2-1 Hirosawa, Wako City, Saitama 351-0198, Japan.
³ Present address: MRC Toxicology Unit, University of Leicester, Lancaster Rd., Leicester LE1 9HN, United Kingdom.
⁴ Present address: Molecular Entomology, Great Lakes Forestry Centre, Canadian Forest Service, 1219 Queen St. East, Sault Ste. Marie, ON P6A5M7, Canada.

	ACKNOWLEDGMENTS

The authors thank Ernst Wimmer for providing the pBac[3xP3-GFPafm] and pBac[3xP3-CFPafm] vectors. We thank Mitsutoshi Ono for technical assistance. This work was supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences of the Bio-Oriented Technology Research Advancement Institution, Japan and grants from the Ministry of Agriculture, Forest and Fisheries and the Ministry of Education, Science, Sports and Culture, Japan.

Manuscript received December 29, 2002; Accepted for publication July 28, 2003.

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