Freshly laid silkworm eggs were microinjected with 1 mgml of any of the three piggybacderived constructs and the helper plasmid pha3pig in equimolar ratio. Pdf flp recombinasemediated sitespecific recombination. Pdf germline transformation of the silkworm bombyx mori. Erratum to germline transformation of the silkworm bombyx. The mobility and transposition functions of piggybac were established and exploited to develop an important system for insect germline transformation 911. Transposon vector systems can possess restricted host ranges 32, 43. Pdf we have developed a system for stable germline transformation in the silkworm bombyx mori l. Injection of the vector and helper plasmid dna into silkworm. Your use of this pdf, the bioone web site, and all posted. The present invention relates to a transgenic silk producing a red fluorescent silk, and more particularly, to a transgenic silk producing a red fluorescent silk which comprises a marker generegulated promoter, a marker gene, a silkwormderived fibroin promoter and an anemone a transformant silk producing a red fluorescent silk can be produced by producing a recombinant expression vector. Characterization of anticd20 monoclonal antibody produced. Nov 01, 2006 read the generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor, biomaterials on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Characterization of anticd20 monoclonal antibody produced by. In addition, it is a key model of the lepidoptera, the second most numerous group of holometabolous insects, which include many beneficials but also the most destructive agricultural pests.
Jan 10, 2007 we investigated the use of minos as a vector for transgenesis in the silkworm, bombyx mori. The intent of these efforts is to create a stable change in the phenotype of the target organism that can be used to answer basic questions about the physiological, genetic, or other effects. Genetic transformation an overview sciencedirect topics. Expression of egfpspider dragline recombination gene in bmn. The ability of an organism to take up dna is called competence. To explore the practical use of transgenic technology in agricultural silkworm varieties, we fused the neomycinresistance gene neo r and the green fluorescent protein gene gfp into the piggybacbased transposon vector and transduced it into silkworms by spermmediated. Research articles germline transformation of the silkworm. Lets explore the different stages of life of a silkworm. One fragment contains the 1st exon and its downstream 1st introns partial sequence. Germline transformation of the silkworm bombyx mori l.
The life history of silkworm moths consists of four distinct stages. The silkworm, bombyx mori, is an agriculturally important insect that has been domesticated for an estimated 5000 years and used extensively for silk production. The silk gland of silkworm is a highly specialized organ that has tremendous ability to synthesize and secrete fibroin. Bombycidae is a completely domesticated lepidopteran insect that has been used for silk production for more than 5000 years international silkworm genome consortium 2008.
The life cycle of the silkworm moth takes about 6 to 8 weeks, depending on temperature. Tamura t, thibert c, royer c, kanda t, abraham e, kamba m, komoto n, thomas jl, mauchamp b, chavancy g, et al. The development of a spider silkmanufacturing process is of great interest. Transgenic silkworms bombyx mori produce recombinant. The life cycle of silk moth starts when a female silk moth lays eggs. The elucidation by oswald avery and colleagues that dna was the transforming agent in streptococcus pneumoniae was one of the.
To explore the practical use of transgenic technology in agricultural silkworm varieties, we fused the neomycinresistance gene neo r and the green fluorescent protein gene gfp into the piggybacbased transposon vector and transduced it into silkworms by. Read the generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor, biomaterials on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. For silkworm germline transformation, preblastoderm nistari embryos were microinjected with a mixture of transgenic plasmid and helper. Genetic transformation in bacteria is the process of taking up free dna from the environment and incorporating it into a recipient cell. In earlier times, use of silk was reserved only for kings and monarchs. Genome engineering and parthenocloning in the silkworm. Eggs hatch after 910 days, larva develop for 2433 days, pupation lasts 814 days and adults live only 34 days. Here, we used the transgenicbased crisprcas9 genome editing system to study the function of the ovo gene in bombyx mori. These eggs are stored over a clean paper or piece of cloth.
However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Germ line transformation of the silkworm, bombyx mori. A transformation system was developed for stable germline transformation in the silkworm bombyx mori l. Moreover, genetic transformation of commercial strains with ox4319 is underway to establish stable transgenic commercial lines by the more direct means. We first constructed a vector plasmid with the green fluorescent protein gfp gene fused with the silkworm cytoplasmic actin gene a3 promoter, and a helper plasmid with the minos transposase gene controlled by the same a3 promoter.
An efficient strategy for producing a stable, replaceable, highly. To achieve efficient expression of rhlf in the middle silk gland of transgenic silkworm, a piggybacbased transgenic vector phsrhlfser1 was constructed fig. In recent years, genetic manipulation technologies have been implemented in the silkworm. Heritable genome editing with crisprcas9 in the silkworm.
For the transgenic crisprcas9 system, the noscas9 line was crossed with the u6sgrna line, and genomic dna was extracted from. Efficient strategies for changing the diapause character of silkworm. Injection of the vector and helper plasmid dna into silkworm eggs. Once a femalespecific lethality system is established in one b. Expression of the hgmcsf in the silk glands of germline. Germline transformation of silkworm was performed as described previously. Its production base is spread over 60 countries with asian countries producing over 90% of mulberry silk production datta and nanavathy 2007. Pdf germline transformation of the silkworm bombyx mori l. A silkworm based silk gland bioreactor for highefficiency. Genetic transformation is the process of integrating exogenous dna into the germline of whole organisms so that it is inherited in subsequent generations. Our results showed that hcl treatment within 3 h of. With the development of genomic and biotechnological tools, b. Germ line transformation of the silkworm, bombyx mori, using.
Despite its importance, genomic research lags behind other models such as drosophila, zebrafish, yeast, and arabidopsis, and a complete genetic variation map was not constructed until 2009. This study represents a direct demonstration of bt resistance gene function in insects with the use of transgenesis. The silkworm bombyx mori is a model organism for lepidopteran research and an important economic species due to the high demand for silk products. We investigated the use of minos as a vector for transgenesis in the silkworm, bombyx mori. The life cycle of a silkworm, from a microscopic egg to a beautiful moth is simply mindblowing.
Germline transformation of the stalkeyed fly, teleopsis. The silkworms feed on mulberry leaves and give rise to pupa. Expression of egfpspider dragline recombination gene in. A piggybacbased transformation vector was constructed that carried spider dragline silk. Sperm mediated transformation insemination of virgin adult females with mixture of linearized dna with semen inherited over three generations combination of this method with transposable elements leads to the stable integration of.
Aug 07, 2004 tamura t, thibert c, royer c, kanda t, abraham e, kamba m, komoto n, thomas jl, mauchamp b, chavancy g, et al. Efficient strategies for changing the diapause character. We confirmed the role of this atpbinding cassette transporter gene in bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. Comparison of transformation efficiency of piggybac. Silkworm the life cycle of silkworm processing of silk. Two different egfphchain fusion genes were expressed in the psg of transgenic silkworms, the products of recombinant proteins were secreted into the psg lumen, aggregated in the msg and spun into cocoons. Excision was detected in the piggybac and the minos assays using both dna and mrna. Expression of the hgmcsf in the silk glands of germline of. Crispr disruption of bmovo resulted in the failure of. Thus, we used piggybac vectors to create transgenic silkworms encoding.
Although somatic sex determination in the silkworm is well characterized, germline sex determination is not. New insight into the mechanism underlying fibroin secretion. Engineering silkworms for resistance to baculovirus. Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation of large amounts of silk from spiders is not feasible. A germline transgenic silkworm that secretes recombinant. Jan 15, 2010 to express human granulocytemacrophage colonystimulating factor hgmcsf gene in the silk glands of transformation silkworm bombyx mori based on genetargeting, two fragments from fibroin heavy chain gene fib h of silkworm were cloned and sequenced. Pcrbased excision assays were used to assay the embryonic activities of three potentially suitable vectors, piggybac, mariner and minos in t. The mixtures of piggybacderived vector and helper plasmid were microinjected into the eggs of three silkworm strains, and the transformation efficiency was compared on the basis of statistical analysis of the. Heatinducible transgenic expression in the silkmoth bombyx mori. In this study, we generated germlinetransgenic silkworms bombyx mori that spun cocoons containing recombinant spider silk. To express human granulocytemacrophage colonystimulating factor hgmcsf gene in the silk glands of transformation silkworm bombyx mori based on genetargeting, two fragments from fibroin heavy chain gene fib h of silkworm were cloned and sequenced. First, we compared the silkworm killing ability of c. We combined transgenic methods and the genomeediting crisprcas9 system to explore the gene function in the silkworm 27,28,29,30. Germline transformation in silkworm piggybac derived construct toshiki et al.
It has extensive prospect of application as a good bioreactor. In pupa stage, a weave is netted around by the silkworm to hold itself. Abstract to overcome the disadvantages of current silkworm bombyx mori transgenic technology, such as costly and time. Quantitative evaluation of cryptococcal pathogenesis and. The generation of germline transgenic silkworms for the.
This study aimed to develop a novel sericin 1 gene ser1 promoterdriven recombinant expression system using transgenic silkworms, in which recombinant proteins are synthesized in msg. For the transgenic crisprcas9 system, the noscas9 line was crossed with the u6sgrna line, and genomic dna was extracted from the noscas9. The caterpillar or larvae are hatched from the eggs of the silk moth. Jan 17, 2012 the development of a spider silkmanufacturing process is of great interest. Silk is a valuable fabric that symbolizes wealth and prosperity. The transposon consists of the piggybac inverted terminal repeats, the enhanced green fluorescent protein gene as the reporter gene and the spider dragline gene.
Targeted gene expression using the gal4uas system in the. We modified the original vector by cloning the coding sequence for bombyx nuclear receptor ftzf1 between the hsp70 promoter and the terminator. Our results showed that hcl treatment within 3 h of oviposition was able to prevent the diapause of eggs. Engineering silkworms for resistance to baculovirus through. A silk thread of the silkworm, bombyx mori, is composed of the insoluble inner fibroin and the hydrophilic outer sericin layer, which are synthesized in the posterior and middle silk gland msg, respectively. Zabelina v, klymenko v, tamura t, doroshenko k, liang h, sezutsu h, et al. The silkworm, bombyx mori, is one of the most economically important insects in many developing countries owing to its largescale cultivation for silk production.
Germline transformation with transposon vectors is an important tool for insect genetics, but progress in developing transformation protocols for butterflies has been limited by high postinjection. The silkworm, bombyx mori, as the foundation of sericulture, is an economically important insect. Pdf flp recombinasemediated sitespecific recombination in. To explore the practical use of transgenic technology in agricultural silkworm varieties, we fused the neomycinresistance gene neor and the green fluorescent protein gene gfp into the piggybacbased transposon vector and transduced it into silkworms by sperm. Efficient strategies for changing the diapause character of. Germline transformation of the butterfly bicyclus anynana. A piggybacderived vector efficiently promotes germline transformation in the silkworm bombyx mori l. The transformation constructs consist of the piggybac inverted terminal repeats flanking a fusion of the b. The transformation method whereby exogenous genes can be introduced into host cells was first established using genetically modified transposons i. The domesticated silkworm is an economically important insect that is widely used as a lepidopteran insect model. The farmers then keep the eggs under the accurate temperature and humidity at a clean place.
Table s1 four silkworm genes and eight associated targeting sites used in this study and. First, we used piggybacmediated germline transformation to generate a transgenic silkworm strain that produces exogenous proteins with high. Transgenebased, femalespecific lethality system for genetic. Important stages in the life cycle of a silkworm animal sake.
A determining factor for insect feeding preference in the. The germline transformation using the piggybac transposon element has also been widely used as a genetic manipulation technology to study gene function in the silkworm 24,25,26. Development of transgenic silkworm for analysis of gene function and productions of recombinant proteinssilks we developed a system for stable germline transformation in the silkworm bombyx mori in 2000. In the beginning, the female silk moth lays hundreds of eggs. Silkworms transformed with chimeric silkwormspider silk. We have developed a system for stable germline transformation in the silkworm bombyx mori l.
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