SERICULTURE

SERICULTURE

WHAT IS SERICULTURE?

INTRODUCTON TO SERICULTURE

HISTORY

DIFFERENT TYPES OF SILK

FEATURES OF INDIAN SILK INDUSTRY

The stages of production OF SILK BY SILK WORM

AN OVER VIEW OF INDIAN SILK INDUSTRYWHERE BIOTECHNOLOGY CAN PLAY A ROLE IN SERICULTURE?Sericulture BiotechnologyMajor Scientific Achievements IN SERICULTURETRANSGENIC SILK WORM

SericultureSericulture is the rearing of silkworms for the production of raw silk.

Although there are several commercial species of silkworms, Bombyx mori is the most widely used and intensively studied.

BOMBYX MORI

Mulberry, Tasar , Eri and Muga.

Tropical tasar silkworm is reared by the tribal inhabitants of Jharkhand, Chattisgarh, Orissa, Andhra Pradesh, Maharashtra and West Bengal States

while the temperate tasar silkworm feeds on oak plants in the North Eastern sub-himalyan states of India. For the extraction of tasar silk three species of Antherea are used in India. They are Antherea mylitta, A. perniyi and A. royeli.

This silkworm is reared on trees of Terminalia tomentosa, Terminalia arjuna. Tasar silkworms are reared wild in nature. They are usually green in colour.

The larvae pass four moults before they complete their duration. Antherea assama produce golden yellow silk. This process is found only in the Brahmaputra Valley of India. It is semidomescticated

This silk produced by Philosamia ricini is called eri silk. It is mostly grown in Assam in eastern parts of India

Muga silkmoth is exclusively found only in Assam state and is known for its unique valuable golden coloured silk fibre.

The non-mulberry silks are called “vanya silks”.

Muga silk+cocoon

Eri silk Tasar silk

Mulbery silk yarn

Silkworm larvae are fed cut-up mulberry leaves, and, after the fourth molt, climb a twig placed near them and spin their silken cocoons.

The silk is a continuous-filament fiber consisting of fibroin protein, secreted from two salivary glands in the head of each larva, and a gum called sericin, which cements the two filaments together.

The sericin is removed by placing the cocoons in hot water, which frees silk filaments and readies them for reeling. The immersion in hot water also kills the silkworm larvae.

The immersion in hot water also kills the silkworm larvae.

Single filaments are combined to form yarn. This yarn is drawn under tension through several guides and wound onto reels.

Finally, the yarn is dried, and the now raw silk is packed according to quality.

The immersion in hot water also kills the silkworm larvae.

Single filaments are combined to form yarn. This yarn is drawn under tension through several guides and wound onto reels.

Finally, the yarn is dried, and the now raw silk is packed according to quality.

The stages of production are as follows:

The silk moth lays eggs.

When the eggs hatch, the caterpillars are fed mulberry leaves.

When the silkworms are about 35 days old, they are 10,000 times heavier than when they hatched. They are now ready to spin a silk cocoon.

The silk is produced in two glands in the silkworm's head and then forced out in liquid form through openings called spinnerets.

The silk solidifies when it comes in contact with the air.The silkworm spins approximately 1 mile of filament and completely encloses itself in a cocoon in about two or three days but due to quality restrictions, the amount of usable silk in each cocoon is small.

As a result, 5500 silkworms are required to produce 1 kg of silk.

The silkworm then metamorphoses and changes into a moth; however, the silkworm is usually killed with heat before it reaches this stage.

The silkworms are killed, because once they reach the moth stage, the moth secretes a fluid to dissolve the silk so it can emerge from the cocoon. This damages the cocoon and the silk then becomes a lower quality.

Some silkworms are allowed to live to be used for breeding

The silk is obtained from the undamaged cocoons by brushing the cocoon to find the outside ends of the filament.

The silk filaments are then wound on a reel. One cocoon contains approximately 1,000 yards of silk filament.

The silk at this stage is known as raw silk. Just one thread consists of 48 individual silk filaments.

A yarn can now be formed by combining several threads of silk.

AN OVER VIEW OF INDIAN SILK INDUSTRY

WHERE BIOTECHNOLOGY CAN PLAY A ROLE IN SERICULTURE?

Sericulture BiotechnologySericulture biotechnology, particularly in India, has assumed significance in recent years,because it provides employment to rural landless and allows utilization of waste and drylands, where nothing can be grown.

In these waste and drylands, mulberry can be grown for silk industrygiving handsome money to the poor marginal farmers.

Mulberry and silkworm constitutethe basic material so that any biotechnology approach should consider improvement of silkworm as well as mulberry

Sericulture is an important industry in India. In the year 1993-94, 13,418 million tonnes of mulberry silk was produced in India However, despite significant progress in sericulture at the global level, Indian sericulture industry is primitive and is not in a position to produce high quality silk in large quantity. due to poor surivivability of these improved strains and a variety of diseases, these improved strains are not very popular.

Other discouraging factors include low precipitation, scanty ground water and poor cultivation practices.

Considerable research at the Central Sericulture Research and Training Institute, (CSRTI), Mysore, is directed towards (i) the improvement of existing mulberry varieties; (ii) introduction of improved mulberry varieties and productive silkworm breeds and (iii) development of silkworm management under tropical conditions. Studies for the production of transgenic silkworm has also been initiated, along with other molecular genetic studies under a collaborative project entitled

Molecular Genetics of Silk Worms: Fundamental Studies and Application to Sericulture.

The project was sanctioned by European Commission and has the following collaborators: Indian Institute of Science, Bangalore (India), Lyon (France), Leuven (Belgium) and Milan (Italy). During 1990-95, the Department ofBiotechnology (Govt. of India) and Central Silk Board (Bangalore) had joint discussions to analyse the constraints of the industry.

They also identified need based priorities in sericulture research and prepared a number of projects to be jointly funded by these two agencies.

Realizing the above, the Department of Biotechnology (DBT) is implementing a programme on application of biotechnology for increasing productivity, enhancing silk quality and improvement of host plants in both mulberry and non-mulberry sericulture.

Major Scientific Achievements Work accomplished on identification and characterization of silkworm microsatellites and utilization of microsatellite derived markers for strain typing, phylogenetics, linkage mapping,

Z-chromosome mapping and analysis of heterologous silkmoths. Application of these markers for identification of productive silkworm hybrids in combination with conventional breeding carried out.

India joined “International Consortium on Lepidopteron Genomics” and fulfilled the Indian commitments viz (a) Construction of high-density linkage map of silkworm (Bombyx mori) (b) Isolation and characterization of ESTs from non-mulberry silkworms (1400 ESTs from tropical tasar, Antheraea mylitta and 35,000 ESTs from muga silkworm, A. assama).

RNA-interference based baculovirus resistant transgenic silkworm lines developed. These lines are undergoing laboratory trials.

A Multi-institutional Network Project on identification of DNA marker for NPV resistance in silkworm has been launched involving CDFD, Hyderabad alongwith other four institutions of Central Silk Board and State Governments.

A collaborative project on identification of DNA markers linked to QTLs (cocoon weight and cocoon shell weight) also launched.

Using BmNPV cell-surface display system, expression of veterinary viral antigens such as fusion glycoprotein (F) of PPRV and hemagglutinin protein (H) of RPV have been achieved on the surface of virions and on the surface of infected cell-lines in cultures as well as infected silkworm larvae (B. mori).

Work on immune response and challenge tests of PPRV antigens produced through Bm NPV expression system in silkworm larvae has been carried out in collaboration with IVRI, Mukteswar.

Microsatellite markers for tasar silkworm and muga silkworms have been developed which are being used to study population structure, gene flow and genetic polymorphisms.

A Multi – institutional project has been launched on phylogeography of Antheraea mylitta (tropical tasar silkworm) and Antheraea assama (muga silkworm). A collaborative project on assessment of genetic diversity of muga silkworm (A. assama) populations in NE region using molecular markers launched.

Characterization of anti-bacterial proteins in silkworm has led to the discovery of unique insect specific lysozyme (similar to C-type lysozyme and alpha-lactalbumins).

A Web-enabled DNA database on mulberry has been developed for the first time in the world encompassing DNA fingerprinting studies carried out on more than 250 genotypes including 50 selected elite genotypes. Efforts have been initiated for construction of a framework molecular linkage map of mulberry for mapping and identification of useful genes.Genetic manipulation of Morus indica cv. K-2 via Agrobacterium mediated transformation using HVA-1 gene for abiotic tolerance has been achieved. Evaluation of putative transgenics in mulberry (with HVA-1 gene) under transgenic green house condition is in progress.

A Multi-institutional Network project has been launched on identification of DNA markers associated with disease and pest resistance in mulberry (Morus spp.) involving CCMB, Hyderabad alongwith four institutions of CSB and state governments.

Goals for 11th Plan

To develop novel high yielding silkworm varieties producing good quality silk and resistant to diseases through marker-assisted breeding and transgenic route.To develop biotic / abiotic stress tolerant mulberry varieties through marker assisted breeding and transgenic route.

To support more basic research in mulberry sericulture (both silkworm and host-plants) including research on disease surveillance and causual agents of major disease of non-mulberry silkworm.

To develop suitalbe technology(s) for by-product utilization in sericulture industry and value addition to the system toward better economic gains.

To develop various applications of silk proteins (both sericin and fibroin) for medical and cosmetic purposes besides using pupa as a source of protein and bio-fuel.

To develop silkworm as bioreactor for producing high-value proteins.

To promote HRD in the area of seribiotecnology by following novel and integrated approaches.

TRANSGENIC SILK WORM

Transgenic silkworms produce recombinant human type III procollagen in cocoons

Collagen is used in many medical applications, including tissue engineering1 and drug delivery materials2, because of its strength and stability as well as its general compatibility with living tissues. Currently, the main source of collagen is cow skin. This source carries a high risk of contamination and can also cause allergic reactions3.

Thus, there is a need for alternative sources of collage to produce large quantities.

The domestic silkworm, Bombyx mori, synthesizes vast amounts of silk protein in its silk glands and spins it into cocoons during the last larval instar.

Recently, a method for stable germline transformation in B. mori was developed using a piggyBac transposon–derived vector4.

B. mori is therefore a good candidate host for the production of recombinant proteins at an industrial scale.

The cDNA of type III collagen is an appropriate choice for a transgene because of its simple gene composition.

To avoid a possible problem with the large size of collagen molecules, we decided to use cDNA of the type III procollagen mini-chain as the actual transgene.

Lees and Bulleid originally designed the procollagen mini-chain which is composed of an N-propeptide, one-fifth of a triple-helix domain, and a C-propeptide5.

In the present study, we produced transgenic silkworms with piggyBac vectors carrying the cDNAs of a fusion protein containing human type III procollagen mini-chains with C-propeptide deleted

The silkworms synthesized the fusion protein in silk glands and secreted it into cocoons. The fusion proteins were purified to a single band on electrophoretic gels

In their cocoons, the insects produced both silk and collagen, which is used to generate artificial skin and cartilage and in cosmetic surgery to fill out lips and wrinkles. The technology has potential to optimize the bulk production of collagen for medical uses.

Abstract

generation of transgenic silkworms that produce cocoons containing recombinant human collagen.

A fusion cDNA was constructed encoding a protein that incorporated a human type III procollagen mini-chain with C-propeptide deleted, a fibroinlight chain (L-chain), and an enhanced green fluorescent protein (EGFP). This cDNA was ligated downstream of the fibroin L-chain promoter and inserted into a piggyBac vector.

Silkworm eggs were injected with the vectors, producing worms displaying EGFP fluorescence in their silk glands. The cocoons emitted EGFP fluorescence, indicating that the promoter and fibroin L-chain cDNAs directed the synthesized products to be secreted into cocoons.

The presence of fusion proteins in cocoons was demonstrated by immunoblotting, collagenase-sensitivity tests, and amino acid sequencing. The fusion proteins from cocoons were purified to a single electrophoretic band. This study demonstrates the viability of transgenic silkworms as a tool for producing useful proteins in bulk.

Transgenic silkworms (Bombyx mori) produce recombinant spider dragline silk in cocoons.

Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, 386-8567, Japan, wenhongxiu@gmail.com.

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. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by the sericin 1 promoter

. Silkworm eggs were injected with the vector, producing transgenic silkworms displaying DsRed fluorescence in their eyes. Genotyping analysis confirmed the integration of the MaSp1 gene into the genome of the transgenic silkworms, and silk protein analysis revealed its expression and secretion in the cocoon.

Compared with wild-type silk, the recombinant silk displayed a higher tensile strength and elasticity. The results indicate the potential for producing recombinant spider silk in transgenic B. mori.

Normal and transgenic adult silkworms (bottom) with red silk glands. ©Nature Biotechnology

Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield. Mol Biol Rep. 2008 Sep; 35(3):329-35. Epub 2007 May 25. [Mol Biol Rep. 2008]Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: Production of recombinant human serum albumin

Application of RNAi for Baculovirus Inhibition

Silkworm is infected by a number of pathogens including the most devastating virus, the B. mori nucleopolyhedrovirus (BmNPV).

As high as 20% of the silkworm cocoon crop is lost due to the BmNPV infection annually in India. The infection is hard to prevent, partly due to sturdy nature of the polyhedra, and partly because of the strains of silkworms.

The strains that are silk yield-wise productive are somewhat more susceptible to the virus.

Development of a viral-resistant strain of the silkworm is thus one of the priorities for sericulturists. With the aim of developing anti-viral strategy, exploited the RNAi to inhibit the expression of an essential viral gene, Immediate-Early-1 (ie-1). This essential viral gene is stimulated early in viral replication and regulates a number of viral genes. To test the efficacy of the RNAi technique in B. mori, they initiated a cell culture based system, in which 420 bp of double stranded RNA to target the 5'-terminal region of IE-1 mRNA (dsie-1), was transfected to two lepidopteran

IE-1 mRNA (dsie-1), was transfected to two lepidopteran insect cell lines, viz. BmN and Sf9. 24 hours post transfection, the cells were infected with BmNPV or Autographa californica multiple nucleopolyhedrovirus (AcNPV), respectively, and western blot was carried out using virus specific anti-gp64 antibody.

The results indicate that there was an initial inhibition of viral proliferation of up to 50% at 24 hours post infection (hpi) as compared to untransfected and virus-infected control cells.

However, by 96 hpi the virus proliferation matched that of the untransfected, but virally infected control cells. To test in vivo efficacy of RNAi, dsie-1 RNA was injected into the hemolymph of silkworm larvae and 24 hours later, the silkworms were infected with BmNPV. Results show that at low level of viral infection, the dsie-1 successfully prevented viral proliferation. However, the high dose of infected viral particles led to larval deaths, comparable to the un-injected, but virally challenged control worms.

For the economically important lepidopteran insect, the silkworm Bombyx mori L, the most devastating viral disease is caused by a nucleopolyhedrovirus, B. mori NucleoPolyhedra Virus (BmNPV).

More than 20% of the crop loss is due to BmNPV infection. With the aim of developing anti-viral strategy, we exploited the RNA interference (RNAi) to inhibit the expression of essential viral genes.In transient cell culture studies, the double stranded RNA (dsRNA) against target baculoviral genes are transfected to BmN cells. The cells are then infected with BmNPV andthe viral inhibition is monitored by western blot.

To develop a strain of silkworm resistant to the baculovirus, transgenic silkworms having transgenes expressing dsRNA against target genes are being produced. PiggyBac mediated transgenesis, is used to integrate the transgene to the silkworm genome. The transgenics are now being tested for their viral resistance.

Multiple genes of BmNPV are also being targeted to enhance the viral resistance.

Baculovirus Inhibition By RNA InterferenceRNA interference (RNAi) is a recently discovered phenomenon of selective mRNA destruction leading to gene knockdown. The first hint of co-suppression was a serendipitous discovery. The unexpected observation of suppression of par-1 gene by both sense and anti-sense strands injected separately to C. elegans was initially noted as a technical aberration. Similarly, a few years later the observation of suggested the probability of RNA induced viral suppression by over expression of sense strand RNA. Apparent influence of dsRNA came to fore by the report of In this study it was shown that when a viral protease gene was expressed either in sense-strand or anti-sense strand, it repressed the viral growth, but the viral inhibition was more profound in the hybrid plants that produced dsRNA. However, it was who showed for the first time that the dsRNA was more potent in inducing gene knockdown than the sense or antisense RNA alone. They suppressed the unc genes of the C. elegans by injecting dsunc RNA. They coined the word RNA interference (RNAi) for this phenomenon. Later they showed that by soaking in dsRNA or by feeding C. elegans with bacteria harbouring dsRNA-producing construct, RNAi could be induced. Subsequently, there has been an explosion of information regarding the RNAi mechanism and the genes knocked down by this technique. RNAi is now believed to be an ancient anti-viral strategy, probably a part of which the cellular machine also utilizes for gene regulation mechanism. For example, Dicer enzyme that is necessary for the initiation of RNAi is also involved in microRNA processing from the stem-loop precursors.

Further, mutation of Arabidopsis homologue of Dicer prevents accumulation of miRNAs. The precursor miRNA transcripts (pre-miRNA) are processed by a RNaseIII nuclease (e.g. Drosha), and are exported from the nucleus (by Exportin-5). Dicer cleaves miRNAs into their mature forms to initiate gene silencing. The 3' two-nucleotide overhang structure has been identified as the specificity determinant in targeting and maintaining small RNAs in the RNAi pathway. Thus the miRNA analyses by genetic and biochemical studies are providing a glimpse at the range of biological processes and phenomena regulated by RNAi. These observations lead us to believe the importance of RNAi not only as a natural weapon against pathogens, but also as a tool for exogenous induction of anti-pathogen strategy. RNAi has been well demonstrated in many arthropod species including the silkworm B. mori

By using PiggyBac mediated transgenesis, we also obtained a transgenic Sf9 cell-line that expresses dsRNA against ie-1 gene. This polyclonal cell-line showed viral inhibition at 48 hpi. These results suggest the possibility of using RNAi in development of baculoviral resistance in the silkworm, B. mori. Using the same construct that gave transgenic Sf9 cell-lines, we have obtained the transgenic silkworms that express dsie-1. These transgenics are made into homozygous line by sib mating and will be used in viral assays. Further investigation is currently being carried out in several of these transgenic silkworms to evaluate the promotion of viral tolerance in the silkworm.

Copyright @ 2008 Silkworms spin medicinal gold By Kate Dalke January 10, 2003Scientists in Japan have genetically modified silkworms to secrete the human protein collagen. In their cocoons, the insects produced both silk and collagen, which is used to generate artificial skin and cartilage and in cosmetic surgery to fill out lips and wrinkles. The technology has potential to optimize the bulk production of collagen for medical uses.

Female silkworm moth depositing eggs. .

This is the first time insects have been used to produce a medically important protein, says Florian Wurm, a biotechnology professor at the Swiss Federal Institute of Technology in Lausanne, Switzerland. Wurm wrote a commentary accompanying the study in Nature Biotechnology.

Currently, most medical collagen comes from cow tissue, which can cause allergic reactions in some patients. Limited quantities of collagen—and other human proteins—are grown in animal cells.

Normal and transgenic adult silkworms (bottom) with red silk glands. ©Nature Biotechnology Japan Science and Technology Corporation in Hiroshima led the project. Seeking an abundant source of patient-friendly collagen, the researchers turned to an industrious insect. They inserted a human gene for collagen into silkworms. The protein was produced in the silk glands and secreted into cocoons, where the collagen was extracted. "The production is extremely fast and it appears that the [extraction] technology is not complicated," says Wurm.

Silkworm production is a major industry in India, Japan, China and Europe, and these resources could be converted to manufacture collagen and other human proteins, the researchers say. They estimate that a facility with 1.5 million silkworms could produce 5 kilograms of collagen per year, in addition to silk. The technology is extremely "low-tech" in the sense that you don't need a molecular biologist to handle the day-to-day manufacturing, says Wurm.

Silkworm cocoons with one cut open to view pupa.

Normal and transgenic adult silkworms (bottom) with red silk glands. ©Nature Biotechnology

Silkworms are larvae of the caterpillar, Bombyx mori, which has been bred for thousands of years for its production of precious fibers. The species is completely domesticated—no wild populations exist. After hatching from eggs, the worms snack on leaves from Mulberry trees and spin their silky cocoons in about three days.Backers of the new biotechnology must still overcome some hurdles. Further research is necessary to determine whether insect cells produce the protein differently from human cells, and the collagen would need to be tested in clinical trials.See related GNN article

Transgenic silkworms produce recombinant human type III procollagen in cocoonsMasahiro Tomita1,4, Hiroto Munetsuna1, Tsutomu Sato1,5, Takahiro Adachi1,2, Rika Hino1, Masahiro Hayashi1,2, Katsuhiko Shimizu1, Namiko Nakamura1, Toshiki Tamura3 & Katsutoshi Yoshizato1,2

We describe the generation of transgenic silkworms that produce cocoons containing recombinant human collagen. A fusion cDNA was constructed encoding a protein that incorporated a human type III procollagen mini-chain with C-propeptide deleted, a fibroin light chain (L-chain), and an enhanced green fluorescent protein (EGFP). This cDNA was ligated downstream of the fibroin L-chain promoter and inserted into a piggyBac vector. Silkworm eggs were injected with the vectors, producing worms displaying EGFP fluorescence in their silk glands. The cocoons emitted EGFP fluorescence, indicating that the promoter and fibroin L-chain cDNAs directed the synthesized products to be secreted into cocoons. The presence of fusion proteins in cocoons was demonstrated by immunoblotting, collagenase-sensitivity tests, and amino acid sequencing. The fusion proteins from cocoons were purified to a single electrophoretic band. This study demonstrates the viability of transgenic silkworms as a tool for producing useful proteins in bulk

IntroductionCollagen is used in many medical applications, including tissue engineering1 and drug delivery materials2, because of its strength and stability as well as its general compatibility with living tissues. Currently, the main source of collagen is cow skin. This source carries a high risk of contamination and can also cause allergic reactions3. Thus, there is a need for alternative sources of collagen to produce large quantities.The domestic silkworm, Bombyx mori, synthesizes vast amounts of silk protein in its silk glands and spins it into cocoons during the last larval instar. Recently, a method for stable germline transformation in B. mori was developed using a piggyBac transposon–derived vector4

. B. mori is therefore a good candidate host for the production of recombinant proteins at an industrial scale. The cDNA of type III collagen is an appropriate choice for a transgene because of its simple gene composition. To avoid a possible problem with the large size of collagen molecules, we decided to use cDNA of the type III procollagen mini-chain as the actual transgene. Lees and Bulleid originally designed the procollagen mini-chain which is composed of an N-propeptide, one-fifth of a triple-helix domain, and a C-propeptide5. In the present study, we produced transgenic silkworms with piggyBac vectors carrying the cDNAs of a fusion protein containing human type III procollagen mini-chains with C-propeptide deleted. The silkworms synthesized the fusion protein in silk glands and secreted it into cocoons. The fusion proteins were purified to a single band on electrophoretic gels

Generation of transgenic silkworms.

WeGeneration of transgenic silkworms.We constructed a cDNA encoding a fusion protein comprising a human type III procollagen mini-chain5, a fibroin L-chain, and EGFP under the control of a fibroin L-chain promoter sequence. This cDNA was incorporated into a piggyBac vector prepared as follows. Preliminary experiments on the expression of this vector in isolated silk glands were performed by transfecting the vector into the glands with a particle gun. These experiments showed that the C-propeptide of the mini-chain strongly suppresses the expression of fusion proteins (data not shown). In the present study we constructed cDNAs containing the sequences of N-propeptide and one-fifth of the triple-helix domain of human type III procollagen (human type III procollagen mini-chain with C-propeptide deleted).We prepared three fusion cDNAs—LE, MOSRA-7, and MOSRA-8—encoding, respectively, fibroin L-chain/EGFP, fibroin L-chain/N-telopeptide/the triple-helix domain of the procollagen mini-chain/C-telopeptide/EGFP, and fibroin L-chain/the C-propeptide-deleted procollagen mini-chain/EGFP (Fig. 1). These cDNAs were inserted between the fibroin L-chain gene 5'-flanking and 3'-flanking sequences; the resulting constructs will be referred to as expression units. The expression units were inserted into pBac[3xP3-DsRed], in which the gene for the red fluorescent protein (DsRed) was introduced as a marker gene under the eye and nervous tissue–specific promoter 3xP3 in place of the EGFP gene of the original piggyBac-derived vector pBac[3xP3-EGFPafm]6. These three vectors were designated pLE, pMOSRA-7, and pMOSRA-8, respectively (Fig. 1).Figure 1: Structures of fusion cDNAs of LE, MOSRA-7, and MOSRA-8 and of the vectors pLE, pMOSRA-7, and pMOSRA-8.