Construction of a piggyBac-based enhancer trap system for the analysis of gene function in silkworm Bombyx mori

Insect Biochemistry and Molecular Biology 38 (2008) 1165–1173

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Construction of a piggyBac-based enhancer trap system for the analysis of gene function in silkworm Bombyx mori Keiro Uchino a,1, Hideki Sezutsu a, *,1, Morikazu Imamura a, b, Isao Kobayashi a, Ken-Ichiro Tatematsu a, Tetsuya Iizuka a, Naoyuki Yonemura a, Kazuei Mita a, Toshiki Tamura a a b

National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan Prion Disease Research Center, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 May 2008 Received in revised form 17 September 2008 Accepted 22 September 2008

Enhancer trapping and insertional mutagenesis are powerful tools for analyzing genetic function. To construct an enhancer trap system in the silkworm Bombyx mori, we developed efficient jumpstarter strains by inserting the piggyBac transposase gene under the control of Bombyx cytoplasmic actin gene (BmA3) promoter into the genome. To stabilize the inserted transgene, the jumpstarter strains were constructed using the Minos transposon as a vector. The ability of each of the 13 jumpstarter strains to remobilize their respective transposons was tested by crossing the jumpstarters with a mutator strain carrying a GAL4 construct containing the BmA3 promoter. Four strains with high remobilization activity were then selected and used to produce enhancer trap lines by crossing with the mutator strains and hybridizing the F1 progeny with a UAS-EGFP strain. Several enhancer trap lines showing characteristic expression patterns at the embryonic, larval, pupal, and adult stages were detected in the subsequent generation. Approximately 10–40% of the silkworms from each cross in the hybridized brood had a remobilized mutator. An analysis of the insertion positions in 105 lines by inverse PCR using a silkworm genome database revealed that remobilization occurred randomly in each chromosome. The frequency of insertion of the remobilized mutator into putative exons, introns, intergenic regions, and repetitive sequences was 12, 9, 36, and 40%, respectively. We concluded that the piggyBac-based GAL4 enhancer trap system developed in this study is applicable for large-scale enhancer trapping in the silkworm. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Bombyx Enhancer trap Mutagenesis piggyBac Silkworm

1. Introduction The silkworm Bombyx mori is used as a model organism to represent lepidopteran insects. A database containing nearly the entire genomic sequence of B. mori was recently constructed (Mita et al., 2004; Xia et al., 2004; Consortium, 2008) and annotation of the genes has already begun (K. Mita, personal communication); however, in order to analyze the function of each annotated gene, a suitable system for studying gene function needs to be developed. To this end, the development of enhancer trapping, insertional mutagenesis, and gene trapping by remobilization of transposons is necessary. Transposon-based enhancer trap systems are useful in the analysis of gene function. Such systems may be used for targeted transgene expression in a stage- and organ/tissue-specific manner. The first enhancer trap system was developed in Drosophila

* Corresponding author. Tel./fax: þ81 29 838 6091. E-mail address: [email protected] (H. Sezutsu). 1 These authors contributed equally to this paper. 0965-1748/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2008.09.009

melanogaster (O’Kane and Gehring, 1987; Cooley et al., 1988), and was successfully used to produce strains with controlled transgene expression (Bellen et al., 1989). Recently, the site of transgene insertion into the chromosome could be determined using the genomic sequence database (FlyBase; http://flybase.bio.indiana. edu/blast/). Insertion of a transgene by transposition disrupts endogenous genes, creating a new mutant gene that can be used to analyze the function of the original gene. Large numbers of enhancer trap and insertion lines constructed in Drosophila (Bellen et al., 2004; Thibault et al., 2004) have successfully been used in post-genomic studies. Notably, the recent development of a similar system in zebrafish (Danio rerio), medaka (Oryzias latipes), red flour beetle (Tribolium castaneum), rice (Oryza sativa), and thale cress (Arabidopsis thaliana) (Ellingsen et al., 2005; Parinov et al., 2004; Lorenzen et al., 2003, 2007; Liu et al., 2005; Ito et al., 2004) has shown that the technology is applicable to multiple species. We previously developed an efficient construction method for transgenic silkworms by the injection of DNA into preblastodermal embryos (Tamura et al., 2000, 2007). The large number of lines required for post-genomic analyses cannot, however, be produced by this technique. Thus, the development of a remobilization

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maintained at Transgenic Silkworm Research Center in National Institute of Agrobiological Sciences (Tsukuba, Japan).

method such as a jumpstart method (Cooley et al., 1988) is desired. In the jumpstart approach, one element (jumpstarter), encoding transposase, efficiently remobilizes the second transposon vector (mutator) by a cross between the jumpstarter and mutator strain. To develop such a system for non-Drosophila insects, Horn et al. (2003) proposed using the piggyBac transposon for the remobilization of a mutator with insertion of the piggyBac transposase gene into a jumpstarter strain via another transposon, such as Minos. In this study, we essentially followed the strategy of Horn et al. (2003) to create an enhancer trap system in Bombyx. The transposon Minos works well as a vector in Bombyx (Shimizu et al., 2000; Uchino et al., 2007), as does the yeast GAL4/UAS system (Imamura et al., 2003). In addition, a construct containing GAL4 under the control of the Bombyx cytoplasmic actin (BmA3) gene may be used as the mutator (Uchino et al., 2006). Using these tools, we attempted to construct an efficient enhancer trap system in silkworm. We first constructed the Minos vector to create a jumpstarter strain. The vector contained the transposase gene of piggyBac transposon under the control of the BmA3 promoter and 3xP3DsRed as a marker. We obtained four highly active jumpstarter strains, which were used to construct enhancer trap lines. We then analyzed their expression patterns and insertion sites. Our results indicate that the enhancer trap system developed in this study can be used to construct large numbers of enhancer trap lines for the analysis of gene function in silkworm. GAL4/UAS approach enables us to control the spatial and temporal expression of a transgene using the enhancer trap lines.

2.2. Construction of the vectors used to create the jumpstarter lines To construct the plasmid for making the jumpstarter strains (pMiBmA3pigTP/3xP3ECFP, Fig. 1A), pMiBmA3pigTP was prepared by inserting the AvaI–BsiWI fragment from pHA3PIG (pHBmA3PIG) (Tamura et al., 2000) into the BsiWI–EcoRI site in pMiA3GFP (pMiBmA3EGFP) (Uchino et al., 2007). 3xP3ECFP was amplified by PCR from pBac3xP3ECFPafm (Horn and Wimmer, 2000) using the primers 50 -AATATGCGAATTCGAGCTCGCCCGGGGATCTAATTC-30 0 and 5 -TAGCCGTACGGTACGCGTATCGATAAGCTTTAAG-30 with LA Taq DNA polymerase (Takara) and then cloned into pGEM-T Easy (Promega). After verification of the sequence, 3xP3ECFP was prepared by digestion with BsiWI and EcoRI and inserted into the BsiWI–EcoRI site of pMiBmA3pigTP. 2.3. Injection of DNA into the embryos Germ-line transformation to construct the jumpstarter strains was performed as described by Uchino et al. (2006). The DNA used for injection (pMiBmA3pigTP/3xP3ECFP) was purified using a Qiagen Plasmid Midi Kit (Qiagen). Transposon Minos mRNA was used as the source of transposase. The vector and mRNA were mixed at a concentration of 0.2 and 0.1 mg/ml, respectively, and injected into preblastodermal w1-pnd embryos. Screening for the transgenic silkworms was performed based on oviposition on day 6–7 using G1 embryos. The transgenic worms were maintained by crossing with strain w1-pnd or w-c.

2. Materials and methods 2.1. Silkworm strains

2.4. Detection of ECFP, EGFP, and DsRed fluorescence

Strain w1-pnd, which is non-diapausing and possesses nonpigmented eggs and eyes, was used to construct the jumpstarter strains. The mutator strain 193-2 (containing the BmA3-GAL4/ 3xP3DsRed construct) and the homozygous UAS-EGFP strain described by Uchino et al. (2006) were used to make the enhancer trap lines. The diapausing strain w-c was used to maintain the silkworms. The silkworm larvae were reared on an artificial diet (Nihon Nosanko) at 25  C. All strains described in this study are

The expression of ECFP from the jumpstarter construct BmA3pigTP/3xP3ECFP, DsRed from the mutator BmA3-GAL4/3xP3DsRed, and EGFP in the enhancer trap lines was detected by fluorescence microscopy MZ16FA (Leica) using CFP, DsRed, and GFP2 filter set, respectively. The images were captured by Leica DFC300FX system (Leica).

A pigTP

BmA3

3xP3ECFP 3‘UTR 1kbp

pMiBmA3pigTP/3xP3ECFP

B

ClaI

ClaI

3xP3DsRed

GAL4

BmA3

Dmhsp70term

C

BmA3-GAL4/3xP3DsRed

Probe

1kbp

UAS-EGFP 1kbp

UAS-EGFP Fig. 1. (A) Physical map of the vector (pMiBmA3pigTP/3xP3ECFP) used to make the jumpstarter strains. (B and C) Illustrations of the BmA3-GAL4/3xP3DsRed and UAS-EGFP mutator constructs: BmA3, promoter region of the Bombyx cytoplasmic actin gene; pigTP, the piggyBac transposase ORF; 30 UTR, the piggyBac transposase 30 UTR; Dmhsp70term, terminal region of hsp70 gene of D. melanogaster; filled arrow, inverted terminal repeat of the Minos transposon; open arrow, right and left arms of the piggyBac transposon; 3xP3ECFP, ECFP with the 3xP3 promoter; 3xP3DsRed, DsRed2 with the 3xP3 promoter; black bar, the plasmid DNA sequence. ClaI fragment (2.4 kb) of BmA3-GAL4/3xP3DsRed was used as a probe for genomic Southern blotting.

K. Uchino et al. / Insect Biochemistry and Molecular Biology 38 (2008) 1165–1173

2.5. Genomic Southern blotting Genomic DNA was isolated from adult moths by SDS-phenol extraction (Ohshima and Suzuki, 1977). 2.4 kb fragment containing GAL4 gene which was derived from ClaI digest of BmA3-GAL4/ 3xP3DsRed (Fig. 1B) was used as probes. 2 mg each of genomic DNA was digested with BglII. The GAL4 probe was hybridized using an Alkphos direct labeling and detection system (GE Healthcare). 2.6. Inverse PCR Genomic DNA was digested with restriction enzymes and then incubated at 70  C for 15 min to deactivate the enzymes. The restriction enzymes, primers, and annealing temperatures used for PCR are listed in Supplementary Table S1. The digested DNA was then purified using a QIAquick PCR Purification Kit (Qiagen) or QIAquick Gel Extraction Kit (Qiagen) and treated overnight with T4 DNA ligase (New England Biolabs) at 4  C. The primers and enzymes were removed using a QIAquick PCR Purification Kit (Qiagen), and the ligated genomic DNA was used as the template for PCR. The first round of PCR was performed using Ex Taq polymerase (Takara) as follows: 94  C for 2 min followed by 35 cycles of 94  C for 30 s, annealing temperature (Table S1) for 30 s, and 72  C 1 min. A second round of PCR was performed using the products from the first round as the template under the same conditions as in the first round of PCR. The amplified fragments were analyzed using an ABI PRISM 3100 genetic analyzer (Applied Biosystems) after cycle sequencing with BigDye Terminator V3.1 (Applied Biosystems) and purification by a DyeEx 2.0 Spin Kit (Qiagen). 2.7. Analysis of the sequences flanking the insertion sites The chromosomal location of the flanking sequences in each enhancer trap line was analyzed using the Bombyx Genome Build2 Sequence Database (KAIKObase; http://sgp.dna.affrc.go.jp/ KAIKObase/). We searched for the nearest gene to the mutator transposon using Bombyx china gene model (Consortium, 2008). In addition, we performed a BLASTN search for the flanking sequences using an EST (KIAKOblast; http://kaikoblast.dna.affrc.go.jp/) and repetitive sequence database (ReAs database; Consortium, 2008). 3. Results 3.1. Construction of the jumpstarter strains and enhancer trap lines We constructed the jumpstarter strains with the plasmid vector pMiBmA3pigTP/3xP3ECFP using the Minos transposon (Fig. 1A). The result of germ-line transformation using the plasmid with Minos transposase mRNA as the source of transposase is summarized in Table 1. Transgenic silkworms carrying the jumpstarter construct BmA3pigTP were detected in 48 G1 broods. From the G1 broods, we initially established 13 jumpstarter strains by crossing with the strain w-c to maintain the lines as diapaused eggs and to use in later experiments. We then measured the ability of the jumpstarter strains to remobilize the mutator. We designed to detect remobilization of the mutator by the change in the EGFP-expression pattern from a BmA3-

Table 1 Construction of jumpstarter strains using pMiBmA3pigTP/3xP3ECFP and Minos transposase mRNA.

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GAL4 original strain (193-2, control) at embryonic and larval stages. Physical maps of the constructs used in our experiments are shown in Fig. 1B and C, while the crossing scheme used to find those silkworms with a remobilized mutator is shown in Fig. 2. The level of activity to remobilize the mutator detected in the G1 females of the different jumpstarter strains is summarized in Table 2. The variable frequency of each EGFP-expression pattern in the insects suggests that the capacity for remobilization varied among the jumpstarter strains. For example, the frequency in jumpstarter strain Js32 was 27.7%, versus 4.7% in strain Js13. Although there was some variation in the frequency of individual crosses, our results indicate that the variation was caused by the strains, suggesting that the establishment of a strain with high remobilization ability is desirable. Higher activity of transposition of P element was observed in the progeny of females compared to that of male parents in Drosophila (Zhang and Spradling, 1993). To compare the remobilizing activity of piggyBac transposon between females and males in silkworm, the level of activity in the G1 males was also studied (Table 2). Specifically, the frequency of each expression pattern among the 13 jumpstarter strains was compared. In contrast to our results for the females, some crosses yielded rather small numbers of G2 eggs. One reason for this result may be the toxicity of GAL4 (Gill and Ptashne, 1988; Habets et al., 2003) in males. Also, weak sterility was observed among the males of the original mutator strain 193-2 (data not shown). Therefore, our frequency values could not be used to directly evaluate the jumpstarter strain males. We also considered the number of fertilized eggs produced in the G2 generation. Based on our criteria, we selected three jumpstarter strains, Js14, Js18, and Js32, for the females and two strains, Js15 and Js18, for the males. We then evaluated the efficiency using the same crossing and detection methods for the selected strains. In the second experiment, at least five repeated crosses were performed for the females (Table 3). The jumpstarter strains Js14, Js18, and Js32 produced an average of 404, 470, and 360 fertilized eggs, respectively. The frequency of enhancer trap silkworms in strains Js14, Js18, and Js32 was 10.7, 14.0, and 41.9%, respectively. To confirm our results for the male jumpstarter strains, we performed two crosses for Js15 and three crosses for Js18 and recorded the number of fertilized eggs produced and the frequency of silkworms with various EGFPexpression patterns (Table 3). Strains Js15 and Js18 laid an average of 456 and 234 fertilized eggs, and the frequency of new enhancer trap silkworms was 29.7 and 14.7%, respectively. Thus, the remobilization ability of the jumpstarter strains listed in Table 2 was confirmed. We next performed Southern blot analysis to examine whether the enhancer trap lines screened by our method possessed the remobilized mutator. Our results clearly show that the position of the band in the insects differed from that in the original mutator strain (Fig. 3), indicating that the mutator transposon in the lines was present in a different position on the chromosome. We next crossed the enhancer trap lines with the diapausing strain w-c and maintained them as diapausing eggs. By repeating the crosses in Fig. 2, we generated more than 200 enhancer trap lines. The rate of production of enhancer trap silkworms in the next generation was 70–80% in our system, and 8–10 new insertions were found in the G3 brood following each cross (data not shown). Unfortunately, those silkworms with strong EGFP expression could not be maintained in the next generation, likely due to the toxicity of GAL4 (Gill and Ptashne, 1988; Habets et al., 2003) in silkworms.

Vector

Number of eggs injected

Number of eggs hatched

Number of G1 brood

Number of positive G1 brood (%)

3.2. Analysis of the enhancer trap lines

pMiBmA3pigTP/ 3xP3ECFP

1097

643

282

48 (17.0)

We first analyzed the expression patterns in our enhancer trap lines. Representative images of the patterns detected are shown in

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Jumpstarter strain

Mutator strain

A

x (BmA3-GAL4,3xP3DsRed / + ; + / + ; + / +)

(+ / + ; BmA3pigTP,3xP3ECFP / + ; + / +)

Selection of the moths with 3xP3DsRed and 3xP3ECFP

x

(BmA3-GAL4,3xP3DsRed / + ; BmA3pigTP,3xP3ECFP / + ; + / +)

UAS-EGFP homozygous strain (+ / + ; + / + ; UAS-EGFP / UAS-EGFP)

Selection of embryos with DsRed and without ECFP in the stemmata

B

Control

Enhancer trap lines

C

Control

Enhancer trap lines

Fig. 2. Crossing scheme used to identify enhancer trap silkworms by the remobilization of BmA3-GAL4/3xP3DsRed (A). Mutator strain 193-2 was used as the BmA3-GAL4 original strain (control). An enhancer trap silkworm was detected by a change in the EGFP-expression pattern in the embryo between days 6 and 8 after oviposition (B) and in the larva at the first molting (C). Complex eye fluorescence in an adult, which was used to detect the presence of the mutator and jumpstarter constructs, is shown. The left and right photos show the expression of DsRed and ECFP, while the letters in parentheses indicate the genotype. DsRed expression in the stemmata of the G2 embryos is indicated by an arrow. The EGFP-expression patterns observed in the G2 embryos and larvae, which were used to detect remobilization, are shown in B and C. Control indicates the expression pattern in the original strain. Mutator strain 193-2 was crossed with a jumpstarter strain containing the BmA3pigTP/3xP3ECFP construct, and those G1 insects with the BmA3-GAL4 and BmA3pigTP constructs were identified at the adult stage based on the expression of DsRed and ECFP in the complex eye. The G1 moths were then mated with the homozygous UASEGFP strain. G2 silkworm embryos expressing DsRed in the stemmata were selected. The EGFP-expression pattern in the selected embryos and larvae was then examined. Those silkworms with a unique EGFP-expression pattern (B and C) were regarded as new enhancer trap silkworms.

Fig. 4. A number of lines exhibited strong expression in the whole larva (Fig. 4A, 1), whereas the pattern of expression in other lines included stripes (Fig. 4A, 3 and 4), mosaic appearances (Fig. 4A, 2 and 7, and B, 1) and specific epidermal cell (Fig. 4A, 8). In addition, several lines exhibited regional expression, including crescent and star marks (Fig. 4A, 5, and B, 2), dermal glands as surface spots (Fig. 4A, 9, and B, 3), and expression in the rectum of posterior region of the larva (Fig. 4A, 6). A number of lines with expression in specific tissues or organs were also studied at the adult stage or by larval dissection. We found lines with relatively specific expression

in larval midgut (Fig. 4C, 1), fat body (Fig. 4C, 2), salivary gland (Fig. 4C, 3) and other tissues. We also found enhancer trap lines with expression in the wing (Fig. 4D, 1–3), antenna (Fig. 3D, 4), silk gland (Fig. 4D, 5), corpus allatum (Fig. 4D, 6), sperm bundle (Fig. 4D, 7), dermal gland (Fig. 4D, 8), and prothoracic gland (Fig. 4D, 9). Since we constructed our enhancer trap lines using the GAL4/UAS system, those lines exhibiting tissue- and organ-specific expression can be used to express other genes. To determine the positions of the insertions in all 105 lines, we sequenced the flanking sequences of the mutator using inverse

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Table 2 Frequency of animals with different EGFP-expression patterns at embryonic and larval stages after crossing mutator with jumpstarter strains. Moths

Jumpstarter strains

Brood number

Number of eggs fertilized

Number of embryos expressing DsRed (A)

Number of embryos and larvae with different EGFP-expression patterns (B)

Frequency of insects with different expression patterns (B/A)%

Female

Js4

1 2 3 1 1 2 3 4 1 2 1 1 2 3 1 1 2

373 159 368 286 425 66 236 398 526 405 423 189 393 452 396 177 504

149 83 142 129 195 34 116 191 226 162 206 50 178 215 166 55 263

23 8 13 6 40 5 14 16 51 20 15 7 14 17 46 7 25

15.4 9.6 9.2 4.7 20.5 14.7 12.1 8.7 22.6 12.3 7.3 14 7.9 7.9 27.7 12.7 9.5

1 1 1 1 1 1 2 1 1 1 1

264 131 152 188 254 101 46 17 275 88 65

Js13 Js14a

Js15 Js18a Js27

Js32a Js45 Male

Js2 Js13 Js14 Js15a Js18a Js29 Js30 Js45 Js46 Js47

a

Av. 300

Av. 281

Av. 194

Av. 345

Av. 341

Av. 74

132 65 65 92 125 51 15 8 114 41 40

Av. 125

Av. 134

Av. 194

Av. 148

Av. 159

Av. 33

Av. 15

Av. 19

Av. 36

Av. 13

Av. 16

6 2 12 6 12 3 0 2 9 11 6

Av. 2

Av. 11.4

Av. 14.0

Av. 17.5

Av. 9.9

Av. 11.1

4.5 3.1 18.5 6.5 9.6 5.9 0 25 7.9 26.8 15

Av. 3.0

These selected strains were analyzed again to test in the second experiment (Table 3).

Table 3 Frequency of animals with different EGFP-expression patterns at embryonic and larval stages in the second experiment. Moths

Jumpstarter strain

Brood number

Number of eggs fertilized

Number of embryos expressing DsRed (A)

Number of embryos and larvae with different EGFP-expression patterns (B)

Frequency of insects with different expression patterns (B/A)%

Female

Js14

1 2 3 4 5 6 7

475 440 524 255 274 466 397

200 173 256 130 130 218 145

33 33 19 8 6 8 28

16.5 19.1 7.4 6.2 4.6 3.7 19.3

Average

404

178

19

10.7

1 2 3 4 5

466 439 552 462 633

139 211 266 224 235

19 41 41 15 34

13.7 19.4 15.4 6.7 14.5

Average

470

215

30

14.0

1 2 3 4 5

551 565 311 200 174

197 206 103 88 88

95 65 34 36 55

48.2 31.6 33.0 40.9 62.5

Average

360

136

57

41.9

1 2

510 409

336 257

99 75

29.5 29.2

Average

459

296

87

29.3

1 2 3 Average

266 359 198 274

106 147 97 116

6 35 12 17

5.7 23.8 12.4 14.7

Js18

Js32

Male

Js15

Js18

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Fig. 3. Southern blotting of genomic DNA obtained from the BmA3-GAL4 transgenic lines. Lane 1, w1-pnd recipient strain (control); lane 2, BmA3-GAL4 original (193-2); lane 3, BmA3-0021; lane 4, BmA3-0013; lane 5, BmA3-0012; lane 6, BmA3-0004; lane 7, BmA3-0014; and lane 8, BmA3-0005.

PCR. The sequence and position of the insertion in the Bombyx genome for each line were analyzed using the Bombyx genome database, EST database and repetitive database (KAIKObase; http:// sgp.dna.affrc.go.jp/KAIKObase/, KIAKOblast; http://kaikoblast.dna. affrc.go.jp/, and ReAs repetitive database; Consortium, 2008). The chromosomal positions, the positions in the sequence scaffold, the distances with the nearest gene, the distances with the nearest repetitive sequence and the putative inserted regions are listed in Supplementary Table S2. Thirty-seven of the insertions were assigned to intergenic regions, while 12 were identified in putative exons (including possible alternative exon), nine were identified in putative introns, and three were found in putative promoter regions. The insertions in the remaining 41 lines were assigned to regions of repetitive sequence; however, the exact site of insertion could not be identified in three lines. All insertions occurred in TTAA sequences, showing that transposition was caused by the piggyBac transposase. The distribution of insertions in each of the chromosomes is shown in Fig. 5. The transposase did not appear to target a particular chromosome; however, chromosome 12 possessed a rather large number of insertions while transposition on chromosome Z occurred only once. Although the site of insertion for mutator original strain 193-2 was located on the fifth chromosome, insertion of the mutator in the enhancer trap lines was detected in almost all of the other chromosomes, suggesting that the prominent local hop observed in transposition systems using the P element in Drosophila (Tower et al., 1993) or piggyBac in Tribolium (Lorenzen et al., 2007) did not occur in our system. Our results are largely in agreement with those produced using piggyBac in Drosophila (Thibault et al., 2004). We also studied the relationship between the insertion position and resulting in strong expression pattern. In some lines, we could see possible relationship between the inserted site and the GAL4 expression pattern. For example, we found that the insertion in the BmA3-0148 strain was occurred in putative intron of BGIBMGA010496 (BmRelA), resulting strong expression in the whole larva (Fig. 4A, 1; Supplementary Fig. S1, A). BmRelA is known as Bombyx NF-kB family proteins controlling antibacterial peptide genes (Tanaka et al., 2005). BmA3-A097, showing patchy expression (Fig. 4A, 2; Supplementary Fig. S1, B), had insertion in putative 50 UTR exon of BGIBMGA011988 (RNP-1). This gene is likely homolog of U1 small nuclear ribonucleoprotein 70 kDa of Drosophila (Mancebo et al., 1990). BmA3-A103 had expression in specific epidermal cell (Fig. 4A, 8; Supplementary Fig. S1, C) and the mutator was found in putative intron of BGIBMGA002581 (ABC transporter). BmA3-0054 showed specific expression in fat body (Fig. 4C, 2), and insertion was occurred in the putative exon of BGIBMGA012940 (undefined). Undefined gene means that the gene has too low similarity with known proteins. BmA3-0070 (Fig. 4D, 3, 9) had mutator in possible promoter region of BGIBMGA002716 (Acyl-CoA oxidase).

Unfortunately, in many lines, the relationship between the insertion site and the expression pattern remains unclear. Representative enhancer trap lines and the nearest genes to the insertion sites were as follows: BmA3-0012 (Fig. 4D, 2), 2.7 kb upstream of BGIBMGA000781 (undefined); BmA3-0050 (Fig. 4D, 5), 6.0 kb upstream of BGIBMGA007694 (Surfeit locus 5); BmA3-0052 (Fig. 4C, 1; 4D, 7), 10 kb downstream of BGIBMGA007429 (Alkaline phosphatase); BmA3-0014 (Fig. 4C, 3; 4D, 6), 16 kb downstream of BGIBMGA010611 (Fatty acid desaturase); BmA3-0004 (Fig. 4A, 6), 14 kb upstream of BGIBMGA011487 (undefined); BmA3-A248 (Fig. 4A, 3), 38 kb upstream of BGIBMGA006302 (undefined); and BmA3-0045 (Fig. 4D, 1, 5, 8), 30 kb upstream of BGIBMGA010174 (undefined). BmA3-201 strain showed very interesting expression pattern in spots (Fig. 4A, 5, Supplementary Fig. S1D), although we could not find any annotated gene at the flanking 120 kb region of the insertion site. Similarly, BmA3-0200 (Fig. 4A, 7) had no annotated gene at the flanking 90 kb region of the insertion site. 4. Discussion In this study, we successfully constructed an enhancer trap system in B. mori using the piggyBac transposon. The piggyBac transposase gene was fused with the Bombyx actin promoter to make jumpstarter strains. Insertion of the piggyBac transposase gene into Bombyx was accomplished using the Minos transposon as a vector. We initially constructed 13 jumpstarter strains and measured their ability to induce the remobilization of the mutator BmA3-GAL4. The remobilization activity of the mutator was measured based on changes in the expression pattern of EGFP at the embryonic and larval stages. In Drosophila, for example in the enhancer trap system of Horn et al. (2003), remobilization of the mutator was detected by the transposition from sex chromosome to autosome; however, the development of a similar method in silkworm was extremely difficult. Of the 28 chromosomes in B. mori, the W (female) chromosome consists of repetitive DNA (Abe et al., 2005), suggesting that the genes on that chromosome are inactivated. Therefore, the use of a line with a mutator-inserted W chromosome as the mutator strain was believed to be impractical. In previous trials, no strain with the transgene inserted into the W chromosome was detected (Uchino et al., 2006; K. Uchino, personal communication). In addition, we cannot use balancer chromosomes that are useful to keep and make homozygote for mutator in Drosophila. Thus, we designed an alternative method to detect remobilization of the mutator. In this study, we measured the remobilization activity of the mutator based on the changes in the expression pattern, so we might underestimate the remobilization frequency. The frequency of mutator remobilization, which varied from less than 10% to 28%, was quite different among the 13 jumpstarter strains (Table 2). We then fixed four strains (Js14, 15, 18 and 32, Table 3) with the ability to induce remobilization at a high frequency and used them to produce new enhancer trap lines. The average maximum frequency of transposition in our system was 41.9% in the G2 embryos when jumpstarter strain Js32 was used. The frequency observed in this study is comparable to that reported for piggyBac-based enhancer detection systems in other insects (Horn et al., 2003; Hacker et al., 2003; Thibault et al., 2004; Lorenzen et al., 2007). Our system successfully produced 8–10 new enhancer trap lines from a single round of mating (data not shown). It may be possible to improve our jumpstarter strains by using a different promoter for expression of the piggyBac transposase gene. Horn et al. (2003) used the Drosophila alpha tubulin promoter to drive the piggyBac transposase gene, but they suggested using the Drosophila hsp70 promoter to produce a more active jumpstarter strain. The Drosophila hsp70 promoter has been applied in Tribolium (Lorenzen et al., 2007), and a high level of activity was obtained. In

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Fig. 4. Representative EGFP-expression patterns in the enhancer trap lines. (A) Dorsal view of a fifth-instar larva. 1, line BmA3-0148; 2, BmA3-A097; 3, BmA3-A248; 4, BmA3-A017; 5, BmA3-0201; 6, BmA3-0004; 7, BmA3-0200; 8, BmA3-A103; and 9, BmA3-A079. (B) Enlarged dorsal view of the fifth-instar larvae in A (lines BmA3-0200[1], BmA3-0201[2], and BmA3-A079 [3]). (C) Organ- and tissue-specific expression in the dissected enhancer trap lines; expressed at midgut and testis (arrow heads) in BmA3-0052 (1), fat body in BmA30054 (2), and salivary glands in BmA3-0014 (3). (D) Organ- and tissue-expression in the BmA3-0045 (1, 4, and 8), BmA3-0012 (2), BmA3-0070 (3 and 9), BmA3-0050 (5), BmA30014 (6), and BmA3-0052 (7) enhancer trap lines. Each picture is of wing (1–3), antenna (4), silk gland (5), corpus allatum indicated by arrow heads (6), sperm bundle (7), dermal gland (8) and prothoracic gland (9).

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U UAS-EGFP Fig. 5. Physical map of the Bombyx genome showing the location of the transposed mutator. The filled triangles, A, and U indicate the positions of the transposed mutator, the position of the mutator in the BmA3-GAL4 original strain (193-2), and the insertion site of the transgene (UAS-EGFP), respectively. Eighty-nine different insertion sites were identified from 105 enhancer trap lines. The insertion site in 16 of the lines was not identified because it occurred in a highly repetitive region. Additional details are provided in Supplementary Table S2.

the future, we plan to use these promoters to create a more effective remobilization system in Bombyx. We obtained many interesting enhancer trap lines showing specific expression in the larval body, wings, silk gland, midgut, fat body, prothoracic gland, corpus allatum, and antennae in this study (Fig. 4). These lines may be useful in analyzing gene function and the expression of foreign genes at specific times and in specific tissues. Indeed, those lines that expressed the transgene in the midgut (Fig. 4C, 1) were successfully used to determine the function of the Y gene (Sakudoh et al., 2007) and densovirus-resistance gene nsd-2 (Ito et al., 2008). However, the expression patterns observed in this study are limited due to the promoter used to create the mutator construct. We previously reported that the core promoter of the Bombyx actin and hsp70 genes did not produce lines with strong expression in our GAL4/UAS system (Uchino et al., 2006). It is possible that the pattern of expression in the enhancer trap lines was affected by the promoter sequence of the mutator. Therefore, we conducted the present experiment in which we constructed an enhancer trap line using a mutator with a different promoter sequence. We found that remobilization of the mutator using the piggyBac transposon resulted in a roughly even distribution of insertion sites on each chromosome (Fig. 5); however, the insertions predominantly occurred in the sequence TTAA. Local hops during remobilization using the piggyBac transposon have been reported in Tribolium but not in Drosophila (Hacker et al., 2003; Thibault et al., 2004; Lorenzen et al., 2007), although the reason is unknown. In this study, most insertions occurred in intergenic or repetitive regions; thus, this may be a general feature of our system. The Bombyx genome is larger than that of Drosophila, and contains a large proportion of repeated sequences (Mita et al., 2004; Xia et al., 2004). The distribution of insertion sites can be explained based on the structure of the Bombyx genome. We could link a possible gene with the observed replace to "EGFP-expression" GFP expression pattern for only a few enhancer trap lines. When the annotation of Bombyx genes progresses, it will be of great help

for understanding the expression patterns. In addition, no lines were identified in which the insertion disrupted a gene of interest. The mutator used in this study tended to cause weak sterility in males, possibly because BmA3-GAL4 have tendency to express in testis (Fig. 4C, 1) and sperm (Fig. 4D, 7). Therefore, we were unable to make homozygotes for mutator transposon to perform insertional mutagenesis experiments using our enhancer trap lines. We have begun to improve mutator transposon and construct a gene trap system to enable insertional mutagenesis. We predict that insertional mutagenesis in Bombyx will be more effective using gene trap system. We are also constructing additional enhancer trap lines and a database so that we may distribute our lines to other scientists. These lines may be more useful in combination with the database developed by Consortium, 2008. Acknowledgments We thank Ms. Sayaka Kobayashi for her technical assistance. We also thank Dr. Toshio Kanda, Mr. Koji Hashimoto and Mr. Kaoru Nakamura for making transgenic silkworms and rearing silkworms, Mr. Michihiko Shimomura for the silkworm genome database analysis. This work was partially supported by the Insect Technology Project of Ministry of Agriculture, Forestry. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.ibmb.2008.09.009. References Abe, H., Mita, K., Yasukochi, Y., Oshiki, T., Shimada, T., 2005. Retrotransposable elements on the W chromosome of the silkworm, Bombyx mori. Cytogenet. Genome Res. 110, 144–151. Bellen, H.J., Levis, R.W., Liao, G., He, Y., Carlson, J.W., Tsang, G., Evans-Holm, M., Hiesinger, P.R., Schulze, K.L., Rubin, G.M., Hoskins, R.A., Spradling, A.C., 2004.

K. Uchino et al. / Insect Biochemistry and Molecular Biology 38 (2008) 1165–1173 The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics 167, 761–781. Bellen, H.J., O’Kane, C.J., Wilson, C., Grossniklaus, U., Pearson, R.K., Gehring, W.J., 1989. P-element-mediated enhancer detection: a versatile method to study development in Drosophila. Genes Dev. 3, 1288–1300. Consortium, 2008. The silkworm genome sequencing. Cooley, L., Kelley, R., Spradling, A., 1988. Insertional mutagenesis of the Drosophila genome with single P elements. Science 239, 1121–1128. Ellingsen, S., Laplante, M.A., Konig, M., Kikuta, H., Furmanek, T., Hoivik, E.A., Becker, T.S., 2005. Large-scale enhancer detection in the zebrafish genome. Development 132, 3799–3811. Gill, G., Ptashne, M., 1988. Negative effect of the transcriptional activator GAL4. Nature 334, 721–724. Habets, P.E., Clout, D.E., Lekanne Deprez, R.H., van Roon, M.A., Moorman, A.F., Christoffels, V.M., 2003. Cardiac expression of Gal4 causes cardiomyopathy in a dose-dependent manner. J. Muscle Res. Cell. Motil. 24 (2–3), 205–209. Hacker, U., Nystedt, S., Barmchi, M.P., Horn, C., Wimmer, E.A., 2003. piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 100, 7720–7725. Horn, C., Wimmer, E.A., 2000. A versatile vector set for animal transgenesis. Dev. Genes Evol. 210 (12), 630–637. Horn, C., Offen, N., Nystedt, S., Hacker, U., Wimmer, E.A., 2003. piggyBac-based insertional mutagenesis and enhancer detection as a tool for functional insect genomics. Genetics 163, 647–661. Imamura, M., Nakai, J., Inoue, S., Quan, G.X., Kanda, T., Tamura, T., 2003. Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori. Genetics 165, 1329–1340. Ito, Y., Eiguchi, M., Kurata, N., 2004. Establishment of an enhancer trap system with Ds and GUS for functional genomics in rice. Mol. Genet. Genomics 271, 639–650. Ito, K., Kidokoro, K., Sezutsu, H., Nohata, J., Yamamoto, K., Kobayashi, I., Uchino, K., Kalyebi, A., Eguchi, R., Hara, W., Tamura, T., Katsuma, S., Shimada, T., Mita, K., Kadono-Okuda, K., 2008. Deletion of a gene encoding an amino acid transporter in the mid gut membrane causes resistance to a Bombyx densovirus (Parvoviridae). Proc. Natl. Acad. Sci. U.S.A. 105 (21), 7523–7527. Liu, P.P., Koizuka, N., Homrichhausen, T.M., Hewitt, J.R., Martin, R.C., Nonogaki, H., 2005. Large-scale screening of Arabidopsis enhancer-trap lines for seed germination-associated genes. Plant J. 41, 936–944. Lorenzen, M.D., Berghammer, A., Brown, S.J., Denell, R.E., Klingler, M., Beeman, R.W., 2003. piggyBac-mediated germline transformation in the beetle Tribolium castaneum. Insect Mol. Biol. 12, 433–440. Lorenzen, M.D., Kimzey, T., Shippy, T.D., Brown, S.J., Denell, R.E., Beeman, R.W., 2007. piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/ helper hybrids. Insect Mol. Biol. 16, 265–275. Mancebo, R., Lo, P.C., Mount, S.M., 1990. Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein. Mol. Cell Biol. 10 (6), 2492–2502. Mita, K., Kasahara, M., Sasaki, S., Nagayasu, Y., Yamada, T., Kanamori, H., Namiki, N., Kitagawa, M., Yamashita, H., Yasukochi, Y., Kadono-Okuda, K., Yamamoto, K., Ajimura, M., Ravikumar, G., Shimomura, M., Nagamura, Y., Shin, I.T., Abe, H., Shimada, T., Morishita, S., Sasaki, T., 2004. The genome sequence of silkworm, Bombyx mori. DNA Res. 11, 27–35. O’Kane, C.J., Gehring, W.J., 1987. Detection in situ of genomic regulatory elements in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 84, 9123–9127.

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Ohshima, Y., Suzuki, Y., 1977. Cloning of the silk fibroin gene and its flanking sequences. Proc. Natl. Acad. Sci. U.S.A. 74, 5363–5367. Parinov, S., Kondrichin, I., Korzh, V., Emelyanov, A., 2004. Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo. Dev. Dyn. 231, 449–459. Sakudoh, T., Sezutsu, H., Nakashima, T., Kobayashi, I., Fujimoto, H., Uchino, K., Banno, Y., Iwano, H., Maekawa, H., Tamura, T., Kataoka, H., Tsuchida, K., 2007. Carotenoid silk coloration is controlled by a carotenoid-binding protein, a product of the Yellow blood gene. Proc. Natl. Acad. Sci. U.S.A. 104, 8941–8946. Shimizu, K., Kamba, M., Sonobe, H., Kanda, T., Klinakis, A.G., Savakis, C., Tamura, T., 2000. Extrachromosomal transposition of the transposable element Minos occurs in embryos of the silkworm Bombyx mori. Insect Mol. Biol. 9, 277–281. Tanaka, H., Yamamoto, M., Moriyama, Y., Yamao, M., Furukawa, S., Sagisaka, A., Nakazawa, H., Mori, H., Yamakawa, M., 2005. A novel Rel protein and shortened isoform that differentially regulate antibacterial peptide genes in the silkworm Bombyx mori. Biochim. Biophys. Acta 1730 (1), 10–21. Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamba, M., Komoto, N., Thomas, J.L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prudhomme, J.C., Couble, P., 2000. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat. Biotechnol. 18, 81–84. Tamura, T., Kuwabara, N., Uchino, K., Kobayashi, I., Kanda, T., 2007. An improved DNA injection method for silkworm eggs drastically increases the efficiency of producing transgenic silkworms. J. Insect Biotech. Sericol. 76, 155–159. Thibault, S.T., Singer, M.A., Miyazaki, W.Y., Milash, B., Dompe, N.A., Singh, C.M., Buchholz, R., Demsky, M., Fawcett, R., Francis-Lang, H.L., Ryner, L., Cheung, L.M., Chong, A., Erickson, C., Fisher, W.W., Greer, K., Hartouni, S.R., Howie, E., Jakkula, L., Joo, D., Killpack, K., Laufer, A., Mazzotta, J., Smith, R.D., Stevens, L.M., Stuber, C., Tan, L.R., Ventura, R., Woo, A., Zakrajsek, I., Zhao, L., Chen, F., Swimmer, C., Kopczynski, C., Duyk, G., Winberg, M.L., Margolis, J., 2004. A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat. Genet. 36, 283–287. Tower, J., Karpen, G.H., Craig, N., Spradling, A.C., 1993. Preferential transposition of Drosophila P elements to nearby chromosomal sites. Genetics 133, 347–359. Uchino, K., Imamura, M., Sezutsu, H., Kobayashi, I., Kojima, K., Kanda, T., Tamura, T., 2006. Evaluating promoter sequences for trapping an enhancer activity in the silkworm Bombyx mori. J. Insect Biotechnol. Sericol. 75, 89–97. Uchino, K., Imamura, M., Shimizu, K., Kanda, T., Tamura, T., 2007. Germ line transformation of the silkworm, Bombyx mori, using the transposable element Minos. Mol. Genet. Genomics 277, 213–220. Xia, Q., Zhou, Z., Lu, C., Cheng, D., Dai, F., Li, B., Zhao, P., Zha, X., Cheng, T., Chai, C., Pan, G., Xu, J., Liu, C., Lin, Y., Qian, J., Hou, Y., Wu, Z., Li, G., Pan, M., Li, C., Shen, Y., Lan, X., Yuan, L., Li, T., Xu, H., Yang, G., Wan, Y., Zhu, Y., Yu, M., Shen, W., Wu, D., Xiang, Z., Yu, J., Wang, J., Li, R., Shi, J., Li, H., Li, G., Su, J., Wang, X., Li, G., Zhang, Z., Wu, Q., Li, J., Zhang, Q., Wei, N., Xu, J., Sun, H., Dong, L., Liu, D., Zhao, S., Zhao, X., Meng, Q., Lan, F., Huang, X., Li, Y., Fang, L., Li, C., Li, D., Sun, Y., Zhang, Z., Yang, Z., Huang, Y., Xi, Y., Qi, Q., He, D., Huang, H., Zhang, X., Wang, Z., Li, W., Cao, Y., Yu, Y., Yu, H., Li, J., Ye, J., Chen, H., Zhou, Y., Liu, B., Wang, J., Ye, J., Ji, H., Li, S., Ni, P., Zhang, J., Zhang, Y., Zheng, H., Mao, B., Wang, W., Ye, C., Li, S., Wang, J., Wong, G.K., Yang, H., 2004. A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science 306, 1937–1940. Zhang, P., Spradling, A.C., 1993. Efficient and dispersed local P element transposition from Drosophila females. Genetics 133, 361–373.