RESUMEN
Microinjection of genetic material into non-diapause eggs is required for genetic engineering of silkworms. Besides diapause could be useful for maintaining transgenic lines, a drawback of this technology is that most standard silkworm strains and experimental lines of interest produce diapausing eggs. Several approaches have been developed to abolish diapause but none are very efficient. Here, we investigated the ablation of the suboesophageal ganglion (SG) in female pupae, which is a source of the hormone required to trigger egg diapause, as a mean to abolish diapause. We showed that SG-ablation is a reliable method to produce nondiapause eggs. Additionally, the challenge associated with lower fecundity of females with SG ablation was resolved by injecting pilocarpine into the mated female. We also investigated the suitability of nondiapause eggs laid by SG-ablated females for transgenesis, targeted mutagenesis, and induction of parthenogenetic development. Our results demonstrated SG-ablation to be a useful and simple method for expanding the possibilities associated with genetic engineering in silkworms.
Asunto(s)
Bombyx , Diapausa de Insecto , Neuropéptidos , Animales , Bombyx/genética , Femenino , Ingeniería Genética , Hormonas , Neuropéptidos/genética , Óvulo , PilocarpinaRESUMEN
The use of parthenogenetic silkworm (Bombyx mori) strains, which eliminate the problem of recombination, is a useful tool for maintaining transgenic clonal lines. The generation of genetically identical individuals is becoming an important tool in genetic engineering, allowing replication of an existing advantageous trait combination without the mixing that occurs during sexual reproduction. Thus, an animal with a particular genetic modification, such as the ability to produce transgenic proteins, can reproduce more rapidly than by natural mating. One obstacle to the widespread use of parthenogenesis in silkworm genetic engineering is the relatively low efficiency of downstream transgenesis techniques. In this work, we seek to optimize the use of transgenesis in conjunction with the production of parthenogenetic individuals. We found that a very important parameter for the introduction of foreign genes into a parthenogenetic strain is the precise timing of embryo microinjection. Our modification of the original method increased the efficiency of transgene injection as well as the survival rate of injected embryos. We also provide a detailed description of the methodological procedure including a graphical overview of the entire protocol.
RESUMEN
Larvae of many lepidopteran species produce a mixture of secretory proteins, known as silk, for building protective shelters and cocoons. Silk consists of a water-insoluble silk filament core produced in the posterior silk gland (PSG) and a sticky hydrophilic coating produced by the middle silk gland (MSG). In Bombyx mori, the fiber core comprises three proteins: heavy chain fibroin (Fib-H), light chain fibroin (Fib-L) and fibrohexamerin (Fhx, previously referred to as P25). To learn more about the role of Fhx, we used transcription activator-like effector nuclease (TALEN) mutagenesis and prepared a homozygous line with a null mutation in the Fhx gene. Our characterization of cocoon morphology and silk quality showed that the mutation had very little effect. However, a detailed inspection of the secretory cells in the posterior silk gland (PSG) of mid-last-instar mutant larvae revealed temporary changes in the morphology of the endoplasmic reticulum. We also observed a morphological difference in fibroin secretory globules stored in the PSG lumen of Fhx mutants, which suggests that their fibroin complexes have a slightly lower solubility. Finally, we performed an LC-MS-based quantitative proteomic analysis comparing mutant and wild-type (wt) cocoon proteins and found a high abundance of a 16 kDa secretory protein likely involved in fibroin solubility. Overall, our study shows that whilst Fhx is dispensable for silk formation, it contributes to the stability of fibroin complexes during intracellular transport and affects the morphology of fibroin secretory globules in the PSG lumen.
Asunto(s)
Bombyx , Fibroínas/genética , Glándulas Salivales , Seda , Animales , Bombyx/genética , Bombyx/ultraestructura , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Fibroínas/metabolismo , Fibroínas/ultraestructura , Mutagénesis Sitio-Dirigida/métodos , Mutación , Glándulas Salivales/citología , Glándulas Salivales/ultraestructura , Seda/química , Seda/genéticaRESUMEN
Clonal transgenic silkworms are useful for the functional analysis of insect genes and for the production of recombinant proteins. Such silkworms have previously been created using an existing ameiotic parthenogenetic strain. However, the process was labor intensive, and the efficiency of producing transgenic silkworms was very low. To overcome this issue, we developed a more convenient and efficient method by breeding non-diapausing parthenogenetic strains. The strains produced non-diapausing eggs only when the embryogenesis of the parent eggs was performed at low temperatures, which could then be used for injecting vector plasmids. This demonstrated that transgenic silkworms could be produced with greater ease and efficiency. To breed the strains, we crossed the existing parthenogenetic strains with bivoltine strains and made F1 and F2 from each cross. Then we selected the silkworms whose eggs have a high ability of parthenogenesis and became non-diapausing. We also demonstrated that the germplasm could be cryopreserved in liquid nitrogen. Thus, this method increases the efficiency and ease of using genetically engineered silkworms to analyze gene function and produce recombinant proteins, potentially impacting various industries.
Asunto(s)
Animales Modificados Genéticamente , Bombyx/genética , Diapausa/genética , Partenogénesis/genética , Animales , Frío , Criopreservación , Desarrollo Embrionario , Genes de Insecto , Ingeniería GenéticaRESUMEN
Lepidopteran insects produce cocoons with unique properties. The cocoons are made of silk produced in the larval tissue silk gland and our understanding of the silk genes is still very limited. Here, we investigated silk genes in the bagworm moth Eumeta variegata, a species that has recently been found to produce extraordinarily strong and tough silk. Using short-read transcriptomic analysis, we identified a partial sequence of the fibroin heavy chain gene and its product was found to have a C-terminal structure that is conserved within nonsaturniid species. This is in accordance with the presence of fibroin light chain/fibrohexamerin genes and it is suggested that the bagworm moth is producing silk composed of fibroin ternary complex. This indicates that the fibroin structure has been evolutionarily conserved longer than previously thought. Other than fibroins we identified candidates for sericin genes, expressed strongly in the middle region of the silk gland and encoding serine-rich proteins, and other silk genes, that are structurally conserved with other lepidopteran homologues. The bagworm moth is thus considered to be producing conventional lepidopteran type of silk. We further found a number of genes expressed in a specific region of the silk gland and some genes showed conserved expression with Bombyx mori counterparts. This is the first study allowing comprehensive silk gene identification and expression analysis in the lepidopteran Psychidae family and should contribute to the understanding of silk gene evolution as well as to the development of novel types of silk.
Asunto(s)
Proteínas de Insectos/genética , Mariposas Nocturnas/genética , Seda/genética , Animales , Evolución Biológica , Bombyx/genética , Fibroínas/genética , Perfilación de la Expresión Génica/métodos , Sericinas/genética , TranscriptomaRESUMEN
ãWe have been constructing a platform for the development of pharmaceutical and medical applications using the domesticated silkworm, Bombyx mori, as a new animal model for drug development and evaluation. Because silkworm larvae originally have the capacity to synthesize up to 0.5 g of silk proteins, genetically modified silkworms (transgenic silkworms) are expected to have high potential in the production of recombinant silks/proteins. An innovative method for generating transgenic silkworms was established in 2000, and ever since this epoch-defining technological development, longstanding efforts have succeeded in developing novel silks that enable the manufacture of new textile materials for regenerative medical uses. Furthermore, we have succeeded in developing a new system of recombinant protein production. This recombinant protein production system is currently capable of producing a maximum of approximately 15 mg recombinant protein per silkworm larva. Transgenic silkworms have also been shown to produce a wide variety of useful proteins, including antibodies and membrane proteins. Some of these recombinant proteins have been in commercial use since 2011. In addition, we have been developing transgenic silkworms as a novel animal model for testing medicines based on metabolic similarities between silkworms and mammals. These applications show the suitability and potential of transgenic silkworms for medical use. Here, we will describe the challenges faced in creating a transgenic silkworm-based platform for pharmaceutical and medical applications.
Asunto(s)
Animales Modificados Genéticamente , Bombyx , Descubrimiento de Drogas , Animales , Descubrimiento de Drogas/métodos , Modelos Animales , Proteínas Recombinantes , Medicina Regenerativa , SedaRESUMEN
The silks produced by caterpillars consist of fibroin proteins that form two core filaments, and sericin proteins that seal filaments into a fiber and conglutinate fibers in the cocoon. Sericin genes are well-known in Bombyx mori (Bombycidae) but have received little attention in other insects. This paper shows that Antheraea yamamai (Saturniidae) contains five sericin genes very different from the three sericin genes of B. mori. In spite of differences, all known sericins are characterized by short exons 1 and 2 (out of 3-12 exons), expression in the middle silk gland section, presence of repeats with high contents of Ser and charged amino acid residues, and secretion as a sticky silk component soluble in hot water. The B. mori sericins represent tentative phylogenetic lineages (I) BmSer1 and orthologs in Saturniidae, (II) BmSer2, and (III) BmSer3 and related sericins of Saturniidae and of the pyralid Galleria mellonella. The lineage (IV) seems to be limited to Saturniidae. Concerted evolution of the sericin genes was apparently associated with gene amplifications as well as gene loses. Differences in the silk fiber morphology indicate that the cocktail of sericins linking the filaments and coating the fiber is modified during spinning. Silks are composite biomaterials of conserved function in spite of great diversity of their composition.
Asunto(s)
Proteínas de Insectos/química , Mariposas Nocturnas/metabolismo , Sericinas/química , Seda/química , Secuencia de Aminoácidos , Animales , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Filogenia , Homología de Secuencia de Aminoácido , Sericinas/genética , Sericinas/metabolismoRESUMEN
For the functional analysis of insect genes as well as for the production of recombinant proteins for biomedical use, clonal transgenic silkworms are very useful. We examined if they could be produced in the parthenogenetic strain that had been maintained for more than 40years as a female line in which embryogenesis is induced with nearly 100% efficiency by a heat shock treatment of unfertilized eggs. All individuals have identical female genotype. Silkworm transgenesis requires injection of the DNA constructs into the non-diapausing eggs at the preblastodermal stage of embryogenesis. Since our parthenogenetic silkworms produce diapausing eggs, diapause programing was eliminated by incubating ovaries of the parthenogenetic strain in standard male larvae. Chorionated eggs were dissected from the implants, activated by the heat shock treatment and injected with the transgene construct. Several transgenic individuals occurred in the daughter generation. Southern blotting analysis of two randomly chosen transgenic lines VTG1 and VTG14 revealed multiple transgene insertions. Insertions found in the parental females were transferred to the next generation without any changes in their sites and copy numbers, suggesting that transgenic silkworms can be maintained as clonal strains with homozygous transgenes. Cryopreservation was developed for the storage of precious genotypes. As shown for the VTG1 and VTG14 lines, larval ovaries can be stored in DMSO at the temperature of liquid nitrogen, transferred to Grace's medium during defrosting, and then implanted into larvae of either sex of the standard silkworm strains C146 and w1-pnd. Chorionated eggs, which developed in the implants, were dissected and activated by the heat shock to obtain females (nearly 100% efficiency) or by a cold shock to induce development to both sexes in 4% of the eggs. It was then possible to establish bisexual lines homozygous for the transgene.
Asunto(s)
Bombyx/genética , Criopreservación/métodos , Genes de Insecto , Animales , Animales Modificados Genéticamente , Desarrollo Embrionario , Femenino , Técnicas de Transferencia de Gen , Respuesta al Choque Térmico , Masculino , PartenogénesisRESUMEN
Silk secreted by the larvae of Hydropsyche angustipennis (Trichoptera) contains serpins HaSerp2A and HaSerp2B that are homologous to serpin 2 known from several lepidopterans and some other insects. The gene HaSerp2A is 2684 bp downstream from the HaSerp2B gene. The genes possess identical exon/intron segmentation (9 exons) and their sequences are nearly identical: only 8 out of 1203 nt differ in the coding region, 4 out of 567 nt in the introns and 2 out of 52 nt in 3' UTR. Both genes are highly expressed in the silk glands whereas expression in larval carcass devoid of the silk glands is hard to detect. Translation products of the genes consist of 401 amino acids, are 98.8% identical, and are secreted as 45 kDa proteins into silk. Homologous genes in similar tandem arrangement occur on chromosome 15 of Bombyx mori (Lepidoptera). The upstream gene BmSerp2B is modified in several exons and does not seem to produce functional mRNA. The gene BmSerp2A contains two copies of exon 9, of which only the second one is used. One kind of mRNA does and the other does not include exon 1, which encodes a signal peptide. The mRNA yielding secreted BmSerp2A is expressed in the posterior, and that encoding the cytoplasmic BmSerp2A in the middle silk gland region; both kinds are strongly expressed in the anterior region. The data indicate that (1) A duplication of serpin 2 gene occurred either before Trichoptera and Lepidoptera diverged as separate orders or independently in early phylogeny of either order; (2) In the caddisfly H. angustipennis, both genes are expressed specifically in the silk glands and generate proteins deposited in the silk; (3) Only one gene seems to be functional in B. mori and is expressed in a cytoplasmic and in a secreted forms in diverse organs, including the silk glands.
Asunto(s)
Proteínas de Insectos/genética , Lepidópteros/genética , Serpinas/genética , Seda/metabolismo , Secuencia de Aminoácidos , Animales , Biblioteca de Genes , Proteínas de Insectos/metabolismo , Lepidópteros/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Serpinas/metabolismoRESUMEN
A binary gene expression system using the yeast GAL4 DNA-binding protein and the upstream activating sequence (UAS) of galactose-driven yeast genes is an established and powerful tool for the analysis of gene function. However, in the domesticated silkworm, Bombyx mori, this system has been limited in its utility by the relatively low transcriptional activation activity of GAL4 and by its toxicity. In this study, we investigated the potential of several established GAL4 variants (GAL4Δ, GAL4VP16, GAL4VPmad2, GAL4VPmad3, and GAL4NFκB) and of two new GAL4 variants, GAL4Rel and GAL4Relish, which contain the transcription-activating regions of the BmRel and BmRelish genes, respectively, to improve the utility of the GAL4/UAS system in B. mori. We generated constructs containing these GAL4 variants under the control of constitutive or inducible promoters and investigated their transcription-activating activity in cultured B. mori cells and embryos and in transgenic silkworms. GAL4VP16 and GAL4NFκB exhibited high transactivation activity but appeared to be toxic when used as transgenes under the control of a constitutive promoter. Similarly, GAL4VPmad2 and GAL4VPmad3 exhibited higher transactivation activity than GAL4, combined with strong toxicity. The transcription-activating activity of GAL4Δ was about twice that of GAL4. The two new GAL4 variants, GAL4Rel and GAL4Relish, were less active than GAL4. Using GAL4VP16 and GAL4NFκB constructs, we have developed a very efficient GAL4/UAS binary gene expression system for use in cultured B. mori cells and embryos and in transgenic silkworms.
Asunto(s)
Bombyx/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Animales , Animales Modificados Genéticamente , Perfilación de la Expresión Génica/métodos , Proteínas Fluorescentes Verdes/genética , Plásmidos/genética , Transcripción Genética , Activación TranscripcionalRESUMEN
To construct an efficient system for the production of recombinant proteins in silkworm (Bombyx mori), we investigated the promoter activity of the silkworm sericin 1, 2, and 3 genes (Ser1, Ser2, and Ser3) using a GAL4/UAS binary gene expression system in transgenic silkworm. The promoter activity of the upstream region of Ser1 was strong, yielding high expression of an enhanced green fluorescent protein (EGFP) transgene in the middle and posterior regions of the middle silk gland (MSG) after day 2 of the fifth instar. The Ser3 upstream region exhibited moderate promoter activity in the anterior MSG, but the Ser2 upstream region did not exhibit any promoter activity. Since the strongest promoter activity was observed for Ser1, we devised a system for the production of recombinant proteins using a GAL4-Ser1 promoter construct (Ser1-GAL4). Transgenic silkworms harboring both the Ser1-GAL4 construct and the previously reported upstream activating sequence (UAS)-EGFP construct, which contains the TATA box region of the Drosophila hsp70 gene, yielded approximately 100 microg EGFP per larva. When we then analyzed the TATA box region, signal peptide, and intron sequences for their effects on production from the UAS-EGFP construct, we found that the optimization of these sequences effectively increased production to an average of 500 microg EGFP protein per transgenic larva. We conclude that this binary system is a useful tool for the mass production of recombinant proteins of biomedical and pharmaceutical interest in silkworm.
Asunto(s)
Animales Modificados Genéticamente/genética , Reactores Biológicos , Bombyx/genética , Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/biosíntesis , Sericinas/genética , Animales , Southern Blotting , Western Blotting , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sericinas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Larvae of the sister orders Trichoptera and Lepidoptera are characterized by silk secretion from a pair of labial glands. In both orders the silk filament consists of heavy (H)- and light (L)-chain fibroins and in Lepidoptera it also includes a P25 glycoprotein. The L-fibroin and H-fibroin genes of Rhyacophila obliterata and Hydropsyche angustipennis caddisflies have exon/intron structuring (seven exons in L-fibroin and two in H-fibroin) similar to that in their counterparts in Lepidoptera. Fibroin cDNAs are also known in Limnephilus decipiens, representing the third caddisfly suborder. Amino acid sequences of deduced L-fibroin proteins and of the terminal H-fibroin regions are about 50% identical among the three caddisfly species but their similarity to lepidopteran fibroins is <25%. Positions of some residues are conserved, including cysteines that were shown to link the L-fibroin and H-fibroin by a disulfide bridge in Lepidoptera. The long internal part of H-fibroins is composed of short motifs arranged in species-specific repeats. They are extremely uniform in R. obliterata. Motifs (SX)(n), GGX, and GPGXX occur in both Trichoptera and Lepidoptera. The trichopteran H-fibroins further contain charged amphiphilic motifs but lack the strings of alanines or alanine-glycine dipeptides that are typical lepidopteran motifs. On the other hand, sequences composed of a motif similar to ERIVAPTVITR surrounded by the (SX)(4-6) strings and modifications of the GRRGWGRRG motif occur in Trichoptera and not in Lepidoptera.
Asunto(s)
Secuencia Conservada/genética , Fibroínas/genética , Genes de Insecto , Proteínas de Insectos/genética , Lepidópteros/genética , Seda/genética , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Insectos/genética , Larva/química , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Bombyx/metabolismo , Regulación de la Expresión Génica/fisiología , Animales , Bombyx/embriología , Bombyx/genética , ADN , Femenino , Marcadores Genéticos , Proteínas Luminiscentes , MasculinoRESUMEN
Silk fiber produced by the larvae of Trichoptera (caddisflies) and Lepidoptera (moths and butterflies) is composed of two filaments embedded in a layer of glue proteins. In an aerial environment Lepidoptera spin silk filaments assembled from heavy chain fibroin (H-fibroin), light chain fibroin (L-fibroin), and the glycoprotein P25. The silk filament of caddisflies, which is produced and persists in water, contained homologues of H-fibroin (>500 kDa) and L-fibroin (25 kDa) but not of P25. The amphiphilic nature of H-fibroin and its high content of charged amino acids probably facilitate the secretion and storage of a covalently linked L-fibroin/H-fibroin dimer in the absence of P25. Several types of short amino acid motifs were arranged in orderly fashion in the regularly reiterated repeats that made up more than 95% of the length of H-fibroin. The H-fibroins of Hydropsyche angustipennis and Limnephilus decipiens from different caddisfly suborders contained GPXGX, SXSXSXSX, and GGX motifs such as the lepidopteran and spider silks but differed from them by a lack of poly(A) and poly(GA) motifs. H-fibroins of both caddisfly species harbored a conserved repeat of 31 residues but were distinguished by a few species-specific motifs and their organization in higher order repeats. Structural differences may be related to the silk function as a catching net in H. angustipennis and a stitching fiber in L. decipiens.
Asunto(s)
Insectos/genética , Larva/fisiología , Lepidópteros/genética , Seda/genética , Seda/ultraestructura , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada , ADN Complementario/genética , Fibroínas/química , Fibroínas/genética , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Seda/aislamiento & purificaciónRESUMEN
The silk of caterpillars is secreted in the labial glands, stored as a gel in their lumen, and converted into a solid filament during spinning. Heavy chain fibroin (H-fibroin), light chain fibroin (L-fibroin), and P25 protein constitute the filament core in a few species that have been analyzed. Identification of these proteins in Yponomeuta evonymella, a moth from a family which diverged from the rest of Lepidoptera about 150 million years ago, reveals that the mode of filament construction is highly conserved. It is proposed that association of the three proteins is suited for long storage of hydrated silk dope and its rapid conversion to filament. Interactions underlying these processes depend on conserved spacing of critical amino acid residues that are dispersed through the L-fibroin and P25 and assembled in the short ends of the H-fibroin molecule. Strength, elasticity, and other physical properties of the filament are determined by simple amino acid motifs arranged in repetitive modules that build up most of the H-fibroin. H-Fibroin synergy with L-fibroin and P25 does not interfere with motif diversification by which the filament acquires new properties. Several types of motifs in complex repeats occur in the silks used for larval cobwebs and pupal cocoons. Restriction of silk use to cocoon construction in some lepidopteran families has been accompanied by simplification of H-fibroin repeats. An extreme deviation of the silk structure occurs in the Saturniidae silkmoths, which possess modified H-fibroin and lack L-fibroin and P25.