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Insect Molecular Biology (2002) 11(3), 217–222 Induction of the white egg 3 mutant phenotype by injection of the double-stranded RNA of the silkworm white gene Blackwell Science, Ltd G. X. Quan, T. Kanda and T. Tamura Insect Biotechnology and Sericology Department, National Institute of Agrobiological Sciences, Ibaraki, Japan Abstract Injection of double-stranded RNA (dsRNA) corresponding to the silkworm white gene (Bmwh3) into preblastoderm eggs of the wild-type silkworm induced phenotypes similar to those observed with mutants of the white egg 3 locus (10 – 19.6). The induced phenotypes were characterized by the presence of white eggs and translucent larval skin. Northern analysis showed that the expression of the endogenous Bmwh3 gene in the injected embryos was distinctly depressed. Furthermore, the injection of the GFP dsRNA inhibited the expression of the GFP gene from a plasmid coinjected with the dsRNA but did not depress the expression of the Bmwh3 gene. These findings demonstrate that sequence-specific RNA interference occurred in the silkworm. We conclude from the results that the RNA interference can be applied as a tool for the analysis of the gene function in the lepidopteran insects. Keywords: Bombyx mori, w3 mutant, white gene, RNAi. Introduction Gene silencing caused by double-stranded RNA (dsRNA), referred to as RNA interference (RNAi), has provided a powerful tool for studying gene function in animals and plants. RNAi was first observed in Caenorhabditis elegans by Fire et al. (1998) who showed that the introduction of dsRNA into the cells inhibited the expression of the corresponding gene. Subsequently, it was found that RNAi occurred in many organisms from fungi to animals and that the method could be applied selectively to knockout gene Received 20 August 2001; accepted after revision 2 January 2002. Correspondence: Toshiki Tamura, Insect Biotechnology and Sericology Department, National Institute of Agrobiological Sciences, Owashi 1 – 2, Tsukuba, Ibaraki 305 – 8634, Japan. Tel.: 81 298386091; Fax: 81 298386028; e-mail: [email protected] © 2002 Royal Entomological Society function. (Sharp, 1999; Misquitta & Paterson, 1999; Catalanotto et al., 2000; Hammond et al., 2000). Selective gene silencing has proven to be highly efficient except in a few cases in zebrafish where silencing was not always sequence-specific (Li et al., 2000; Oates et al., 2000; Zhao et al., 2001). The mechanism of gene silencing by dsRNA results from cleavage of the dsRNA into fragments of about 22 nucleotides within the cells (Zamore et al., 2000; Elbashir et al., 2001). These nucleotide fragments serve as guide sequences that assemble a silencing complex to destroy specific messenger RNAs (Zamore et al., 2000; Bernstein et al., 2001; Hammond et al., 2001). RNAi has been utilized with some success to assess activity of specific genes in Drosophila (Kennerdell & Carthew, 1998; Bhat et al., 1999; Misquitta & Paterson, 1999; Willert et al., 1999; Yu et al., 1999; Huang et al., 2000). These investigations resulted in silencing of embryonic genes after injecting dsRNA into syncytial blastoderm-stage embryos. RNAi has been used successfully in silencing embryonic genes in the red flour beetle, Tribolium castaneum (Brown et al., 1999). It was also effective in silencing larval haemocyte genes in the flesh fly Sarcophaga (Nishikawa & Natori, 2001). However, efforts to silence late-acting genes using dsRNA injected into early embryos have met with little success due to dilution from progressive cell divisions. RNAi for the Drosophila white eye gene resulted in recovery of less than 3% of knockouts in adults (Misquitta & Paterson, 1999). For most non-dipteran insects, RNAi has not been assessed as a tool for the silencing of specific genes. To investigate whether the method can be applied to lepidopteran insects, we examined the effect of dsRNA on the silkworm Bombyx mori white gene (Bmwh3 ). The Bmwh3 gene has been reported to be homologous to the Drosophila white gene (Abraham et al., 2000). The Drosophila WHITE is involved in the transport of the precursors of the ommochrome and pteridine pigments (Sullivan et al., 1980; Ewart et al., 1994; Mackenzie et al., 1999) in the eye where they combine to produce the distinctive eye colour. Mutants of the white gene lose the ability to transport the precursors and fail to accumulate the pigments in the eye (Mackenzie et al., 1999), resulting in the white eye phenotype in Drosophila. 217 218 G. X. Quan, T. Kanda and T. Tamura The Bmwh3 gene is a suitable gene to determine the effect of the dsRNA-mediated interference in the silkworm. In the wild-type silkworm, the eggs and the eyes are dark brown. This colour is caused by the accumulation of ommochrome pigments. Although several different loci responsible for the white egg mutations have been identified, the silkworm white (Bmwh3 ) gene is related to mutations of the white egg 3 (w3 ) locus (10 – 19.6) (Abraham et al., 2000). The w3 locus is multiallelic, and several mutations, such as the white egg 3 (w3), the white-egg translucent (w3 oe ) and Aojuku white-egg translucent (w3 ol ) have been mapped to this locus. Currently RNAi is most effective on genes expressed during the embryonic and early larval stages, and the presence of a few dsRNA molecules per cell is sufficient to silence gene expression (Fire et al., 1998; Kennerdell & Carthew, 1998). This fact indicates that genes related to egg colour mutants would be the most suitable genes to determine whether the RNAi acts in the silkworm because these genes are expressed at the early stage of the embryonic development and the morphological changes can be easily observed. In addition, the function of the Bmwh3 gene is cell-autonomous, and the protein controls the transport of the precursor of the ommochrome pigment. It is expected that the silencing of this gene should directly induce morphological changes. Therefore, the Bmwh3 gene appears to be a good candidate to test gene silencing by RNAi in the silkworm. In this work, we assessed the effect of injection of Bmwh3 dsRNA into preblastoderm embryos of B. mori to establish the utility of RNAi in Lepidoptera. Injection of dsRNA corresponding to Bmwh3 gene induced phenotypic white eggs and translucent skinned larvae. The amount of Bmwh3 transcript was concomitantly reduced. Furthermore, injection of dsRNA corresponding to the GFP gene did not induce the white egg and translucent larval phenotype, suggesting that inhibition of the gene by the dsRNA was sequence specific. Results Effects of the injection of the Bmwh3 dsRNA on the egg and larval phenotype To determine the effect of dsRNA on the silkworm white gene (Bmwh3) (Fig. 1A), we injected dsRNA corresponding to Bmwh3 into the wild-type preblastoderm embryos. Eggs of the wild-type silkworm are generally white just after oviposition and the egg colour turns dark brown as embryonic development progresses (Fig. 2A). Immediately after hatching, the cuticle of the larvae is dark brown. The cuticle changes to white and opaque within 2 days after hatching. The phenotypes of the mutations of the w3 locus were slightly different in each mutant strain. For example, the eggs of the w3 mutant were light brown (Fig. 2A) and the larval skin was semi-translucent (Fig. 2A). In the w3 ol and the w3 oe mutants, the eggs were white and the larval skin was highly translucent (Fig. 2A). Other mutants for the w3 allele exhibited similar morphological characters. Therefore, the mutants belonging to this group were generally characterized by the presence of white eggs and of larvae with translucent skin. If these characters are related to the gene function of the Bmwh3 as suggested by Abraham et al. (2000), the depression of the Bmwh3 gene should result in Figure 1. Features and sources of dsRNA used in the experiment. (A) Representation of the structure of the silkworm Bmwh3 cDNA. The 925 bp dsRNA of the Bmwh3 gene is denoted by double lines. The probe used for the Northern analysis is indicated by the striped box. (B) Structure of the plasmid pPIGA3GFP. The 706 bp dsRNA of the GFP gene is denoted by double lines. © 2002 Royal Entomological Society, Insect Molecular Biology, 11, 217– 222 Induction of w3 phenotype by dsRNA 219 Figure 2. Phenotypic changes following injection of the Bmwh3 dsRNA into the wild-type eggs. The black and white bars in the photo indicate 2 mm and 0.2 mm, respectively. (A) Eggs and larvae of the wild-type strain 200 and the mutants w3, w3 ol and w3 oe. (B) Induction of the white eggs and translucent larvae by the injection of the Bmwh3 dsRNA. The injection of the Bmwh3 or GFP dsRNA was performed in the eggs of the wild-type silkworm 200 at the preblastoderm stage. The egg colour was observed at 4 days after oviposition and the larvae were observed on the third day of the first instar. (C) Expression of the GFP gene injected into the eggs. The plasmid pPIGA3GFP (0.20 µg /µl) alone or with either the GFP (10 nM) or Bmwh3 (10 nM) dsRNAs was injected into the eggs. The expression of the GFP gene was observed on the 4th day after oviposition. © 2002 Royal Entomological Society, Insect Molecular Biology, 11, 217– 222 220 G. X. Quan, T. Kanda and T. Tamura Table 1. Effect of the dsRNA injected into the eggs on the phenotypic changes Injection Concentration of DsRNA* (nM) Number of injected eggs Number of developed eggs Number of eggs with the following colour (%) Dark brown Mosaic White Number of eggs with GFP expression(%) Number of hatched larva (%) Number of translucent larvae (%) DsRNA of GFP 0.1 1 10 100 51 50 50 51 46 45 46 48 46(100) 41(91) 46(100) 48(100) 0(0) 4(9) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) n n n n 7(15) 9(20) 14(30) 10(21) 0(0) 0(0) 0(0) 0(0) DsRNA of Bmwh3 0.1 1 10 100 96 99 104 102 92 99 95 100 86(94) 69(70) 12(13) 1(1) 4(4) 29(29) 51(54) 16(16) 2(2) 1(1) 32(34) 83(83) n n n n 26(28) 19(19) 14(15) 25(25) 0(0) 0(0) 0(0) 5(20) 0 10 10 50 51 50 50 47 48 46(92) 41(87) 0(0) 3(6) 6(13) 5(10) 1(2) 0(0) 43(90) P P + GFP P + Bmwh3 45(90) 0(0) 36(75) n n n n n n *Injection of 2 – 3 nL of 0.1 – 100 nM solution of the dsRNA, corresponding to roughly 105−108 molecules; P, GFP plasmid DNA (0.20 µg/µL); P + GFP, GFP plasmid DNA (0.2 µg/µL) and 10 nM dsRNA of GFP gene; P + Bmwh3, GFP plasmid DNA (0.2 µg /µL) and 10 nM dsRNA of Bmwh3; n, not examined. the induction of white egg and of larvae with translucent skin. Injection of dsRNA for Bmwh3 induced white eggs as well as brown and white mosaic eggs as expected (Fig. 2B). On the other hand, eggs injected with GFP dsRNA as the non-specific control were dark brown (Fig. 2B). The frequency of the appearance of the white and mosaic eggs varied depending on the amount of Bmwh3 dsRNA injected. The greater the concentration of the dsRNA solution injected, the higher the frequency of appearance of white and mosaic eggs (Table 1). When a 100 nM solution of Bmwh3 dsRNA was injected, most of the eggs became white. It was only at the 100 nM concentration of Bmwh3 dsRNA that larvae exhibited a phenotype with a translucent skin (Fig. 2B, Table 1). Although the frequency of larvae exhibiting translucent skin was not high, those larvae with the phenotype maintained the condition until the second instar. Injection of the GFP dsRNA inhibits only the expression of the GFP gene Figure 3. Northern blot hybridization of the RNAs extracted from the eggs injected with the dsRNA of the Bmwh3 gene. The Bmwh3 dsRNA (100 nM) or the GFP dsRNA (100 nM) were injected into the eggs and the RNAs were collected from the eggs at 4 days after oviposition. The eggs without injection corresponded to the control. Eight micrograms of the purified + poly(A) RNAs were used in each lane and hybridized with the probe of the Bmwh3 gene (Fig. 1A) or with that of the silkworm elongation factor 1-α. To determine whether the dsRNA silencing was sequence specific, we examined the effect of dsRNA on the expression of the Bmwh3 and GFP genes in embryos. Microinjection of the plasmid pPIGA3GFP into preblastoderm embryos results in transient expression of the GFP gene in 90% of the eggs (Table 1, Fig. 2C). On the other hand, co-injection of the pPIGA3GFP plasmid with GFP dsRNA eliminated GFP expression in all of the eggs injected and the eggs had wild-type brown colouration. Co-injection of the pPIGA3GFP plasmid with Bmwh3 dsRNA resulted in more than 70% of the eggs showing GFP expression. In addition, many eggs exhibited the white or mosaic phenotype indicating that the dsRNA of the Bmwh3 gene silenced only the expression of the Bmwh3 gene. These results indicate that interference of © 2002 Royal Entomological Society, Insect Molecular Biology, 11, 217– 222 Induction of w3 phenotype by dsRNA the dsRNAs in the silkworm occurred in a sequencespecific manner. DsRNA depresses levels of the corresponding mRNA A Northern analysis was performed to assess the effect of dsRNA silencing on the levels of specific RNAs in the injected eggs. Poly(A)+ RNA was isolated from each group of eggs and equal amounts of poly(A)+ RNA were electrophoresed for each preparation. Uninjected eggs and eggs injected with GFP dsRNA contained significant amounts of endogenous Bmwh3 mRNA (Fig. 3). However, eggs injected with Bmwh3 dsRNA contained a much smaller amount of mRNA when compared with the control eggs. Equal loading of the samples was confirmed by hybridization with a probe for the silkworm EF1-α. This finding suggests that the white egg and translucent skin larvae phenotypes observed after injection of Bmwh3 dsRNA was due to a reduction in the amount of Bmwh3 mRNA and consequently a reduction in the accumulation of pigments. Discussion We have shown that injection of Bmwh3 dsRNA into preblastoderm eggs caused silencing of the Bmwh3 locus and induction of the white eggs and translucent larvae phenotypes similar to that observed in white egg 3 (w3) mutations. The action of the Bmwh3 dsRNA resulted in a reduction in the level of Bmwh3 mRNA accumulating in the eggs. This effect was sequence specific because injection of GFP dsRNA did not reduce endogenous Bmwh3 mRNA. Sequence-specific silencing was also shown for GFP expression by interfering with the transient expression of GFP from a plasmid without affecting the colour of the eggs and larvae. This is the first report in which it is shown that RNAi acts in the silkworm. We conclude from these results that the mechanism of sequence-specific degradation by dsRNA exists in the silkworm and that the injection of dsRNA into eggs can be used to analyse gene function in the silkworm, especially for the genes expressed at the early embryonic stage. Application of this technique to gene function analysis is critical to the examination of more than 24000 ESTs of the silkworm genes which are available as a data base (SilkBase, http://www.ab.a.u-tokyo.ac.jp/silkbase/). We expect that the method established in these experiments will be applied to further analyse the gene function of the silkworm and contribute to the progress of post-genome studies in lepidopteran insects. 221 colour of the strain 200 is dark brown and the larval skin is white and opaque. The mutant strains for the white egg 3 allele, e01 ol white egg 3 (w3 ), e02 Aojuku white-egg translucent (w3 ) and e03 oe white-egg translucent (w3 ) were obtained from the Institute of Genetic Resources, Kyushu University, Kyushu, Japan. The larvae were reared on an artificial diet (Nihon Nosan) at 25 °C. To break the embryonic diapause, eggs within 4 h after oviposition were treated with 0.9 N HCl for 1 h at 25 °C. The eggs were kept until 6 – 7 h after oviposition and were then used for the microinjection of dsRNA by the method of Kanda & Tamura, 1991. Two to three nl of the solution were injected into each egg. The injected eggs were incubated at 25 °C in a moist Petri dish. Eggs that were viable after four days of embryonic development were observed for the expression of GFP using a fluorescence microscope with the GFP2 filter (Leica). Construction of the dsRNAs To produce the dsRNAs for Bmwh3 and GFP, PCR fragments for each were cloned into pGEM-T plasmid (Promega). A 925 bp DNA fragment of the silkworm white gene (Bmwh3, Abraham et al., 2000), was amplified using Bmwh3 cDNA as template with the primers CTACGGAGCCATCGGAGGTATT and GGCCGTGCAAACTGTATCTATG. A 706 bp DNA fragment of the green fluorescent protein (GFP) gene was PCR amplified using the plasmid pPIGA3GFP (Tamura et al., 2000) as the template with the primers TGGTGAGCAAGGGCGAGGAG and TCGTCCATGCCGAGAGTGAT. Plasmids with PCR fragments inserted in both directions for the Bmwh3 and GFP genes were isolated for the synthesis of the dsRNAs. The sense and antisense RNAs were synthesized for each gene target using the RiboMAX Large Scale RNA Production Systems-T7 (Promega). The plasmids were linearized by the digestion with the restriction enzyme PstI and used as the template for the RNA synthesis. Following the RNA synthesis, the reaction mix was extracted with phenol / chloroform, precipitated with ethanol and dissolved in RNase-free water. Equal molar amounts of the sense and antisense RNAs were mixed in the 10 mM Tris-HCl (pH 7. 5) / 20 mM NaCl buffer, and heated at 95 °C for 1 min. To anneal the dsRNA, the heated RNA solution was kept at 25 °C for 12–16 h. The annealed dsRNA was treated with RNaseA (1 µg/ml) at 37 °C for 30 min, extracted with phenol/ chloroform, precipitated with ethanol and dissolved in distilled water. The formation of the dsRNA was confirmed by non-denaturing agarose gel electrophoresis. The injection solutions were prepared by dilution of the dsRNAs in distilled water. Northern blot hybridization Total RNA was purified by ISOGEN (Nippongene) from developing eggs four days after oviposition for Northern blot hybridization. Poly(A)+ RNA was isolated with Oligotex-dT30 <Super> (Roche Molecular Biochemicals). The poly(A)+ RNAs were separated on a 1% agarose gel by the method of Goda & Minton (1995) and transferred to a Hybond-N membrane (Amersham Pharmacia Biotech). Hybridization and detection were performed with the AlkPhos Direct Labeling and Detection System (Amersham Pharmacia Biotech). Experimental procedures Silkworm strains and microinjection The bivoltine silkworm strain 200 was obtained from the Gunma Sericultural Experiment Station and used as a wild-type. The egg- Acknowledgements We thank Dr N. Kômoto (Genetics and Evolution Department, National Institute of Agrobiological Sciences) for © 2002 Royal Entomological Society, Insect Molecular Biology, 11, 217– 222 222 G. X. Quan, T. Kanda and T. Tamura useful discussions and Dr P. Shirk (USDA-ARS) for critical reading of the manuscript. 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(2001) Double-stranded RNA injection produces nonspecific defects in zebrafish. Dev Biol 229: 215 – 223. © 2002 Royal Entomological Society, Insect Molecular Biology, 11, 217– 222