Title of Invention	METHOD FOR PRODUCING TRANSGENIC SILKWORM THAT SHOWS RESISTANCE AGAINST NUCLEAR POLYHEDROSIS VIRUSES
Abstract	An objective of the present invention is to provide transgenic silkworms that show resistance against NPV, and methods for producing them. Transgenic silkworms carrying an expressive DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of NPV were produced. Studies on NPV proliferation in such transgenic silkworms showed that proliferation of the virus can be suppressed.

Title of Invention

METHOD FOR PRODUCING TRANSGENIC SILKWORM THAT SHOWS RESISTANCE AGAINST NUCLEAR POLYHEDROSIS VIRUSES

Abstract

An objective of the present invention is to provide transgenic silkworms that show resistance against NPV, and methods for producing them. Transgenic silkworms carrying an expressive DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of NPV were produced. Studies on NPV proliferation in such transgenic silkworms showed that proliferation of the virus can be suppressed.

Full Text	TRANSGENIC SILKWORMS THAT SHOW RESISTANCE AGAINST NUCLEAR POLYHEDROSIS VIRUSES, AND METHODS FOR PRODUCING THEM FIELD OF THE INVENTION The present invention relates to transgenic silkworms that show resistance against nuclear polyhedrosis viruses. Furthermore, this invention relates to methods for producing transgenic silkworms that show resistance against nuclear polyhedrosis viruses. BACKGROUND OF THE INVENTION At the work site of sericultural industry, silkworms are seriously damaged by nuclear polyhedrosis viruses (NPV). To date, many researchers have worked on the production of NPV-resistant silkworms, but, to date, no one has yet succeeded. This is due to the fact that the gene for resistance against NPV is absent in all silkworms preserved in the world. To prevent damages from these viruses, thorough sterilization of the silkworm incubation areas and related facilities has been encouraged. However, for sericulture farms, such treatment is not only troublesome but also involves tremendous labor and resource costs, makes cocoons costly, and is one of the factors complicating farming itself. NPV is a large DNA-type virus that carries at least 50 or more genes. It is known to proliferate and express these genes in host silkworm cells. A number of these genes are known to be essential for viral growth (Kool M, Ahrens CHS Goldbach RW, Rohnnann GF5 Vlak JM. (1994) Identification of genes involved in DNA replication of the Autographa califoraica baculovirus. Proc.Natl. Acad. SciUSA91(23): 11212-11216; LuA, Miller IX (1995) The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication. J. Virol 69(2): 975-982). Recently, new methods for producing transgenic silkworms, utilizing transposons or marker genes, have been developed (Tamura, T. (1999) Methods for producing transformed silkworms using transposons. Abstracts of the 7th Conference on Insect Functions, pi 0-22; Horn, C, B. Jaunich and E. A. Wimmer, (2000) Highly sensitive, fluorescent transformation marker for Drosophila transgenesis, Dev Genes Evol 210: 623-629; Horn, C.? and E. A, Wimmer, (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210: 630-637; Thomas, J. L., M. Da Rocha, A. Besse, B. Mauchamp and G Chavancy, 2002 3xP3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the embryonic stage onwards. Insect Biochem Mol Biol 32:247-253; Tamura, T., Thibert, C., Royer, C, Kanda, X, Abraham, Es Kamba, M, Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G, Shirk, P., Fraser, M, Prudhomme, J.-C. and Couble, P. (2000) A piggyBac element-derived vector efficiently promotes germ-line transformation in the silkworm Bombyx mori L. Nature Biotechnology 18, 81-84; Berghammer, I A. J., M. Klingler and E. A. Wimmer, (1999) A universal marker for transgenic insects. Nature 402: 370-371; Tomita, M., Sato, T., Adachi,15 Munetsuna, H., Tamura, T., Kanda, T.f Yoshizato, K, (2001) Production of human collagen gene-incorporated transgenic silkworms. Abstracts of the 24th Annual Meeting of the Molecular Biology Society of Japan). The methods use DNA-type piggyBac transposon as the vector, and the green fluorescent protein (GFP) gene as the marker gene. Since these methods can efficiently produce recombinants, they may be used under various practical situations, such as production of useful proteins (Tomita, M.„ M. H., Sato T, Adachi T, Hino R, Hayashi M, Shimizu K, Nakamura N, Tamura T, Yoshizato a K.s (2003) Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nat BiotechnoL 21,52-56.) and new fibers, and development of varieties that are granted resistance against viruses. Accordingly, parts of such studies have been undertaken. SUMMARY OF THE INVENTION The present invention was made in view of the above-mentioned circumstances. An objective of the present invention is to provide transgenic silkworms showing resistance against NPV, and methods for producing them. More specifically, the objective is to provide NPV-resistant transgenic silkworms which carry an expressive DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for NPV proliferation, and methods for producing such silkworms. The present inventors carried out extensive analyses to achieve the above-mentioned objective. First, the inventors examined whether NPV-resistant silkworms could be produced by utilizing transgenic silkworms. More specifically, a gene that forms an mRNA with a hairpin-type structure was artificially produced utilizing a virus-derived gene as the template, and NPV proliferation in transgenic silkworms carrying this gene was investigated. As a result, proliferation of viruses was found to be significantly suppressed. More specifically, the present invention relates to transgenic silkworms showing resistance against NPV and methods for producing them. Specific objectives of the present invention, objects [1] to [17], are described below. [1] A transgenic silkworm that shows resistance against nuclear polyhedrosis viruses wherein the silkworm carries an expressive DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses. [2] The transgenic silkworm of [1], wherein the DNA encoding an RNA molecule is a DNA encoding an RNA molecule with a hairpin-type structure, [3] The transgenic silkworm of [2], wherein the DNA encoding an RNA molecule with a hairpin-type structure is a DNA in which a sense code DNA encoding a sense RNA of any one of the regions of an mRNA encoding a gene essential for proliferation of nuclear polyhedrosis viruses, and a DNA complementary to said sense code DNA are linked through a linker such that the sense code DNA and complementary DNA face each other. [4] The transgenic silkworm of any one of [1] to [3], wherein the gene essential for proliferation of nuclear polyhedrosis viruses is the lefl gene. [5] The transgenic silkworm of [4], wherein the DNA encoding a sense RNA is the DNA of (a) or(b): (a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1; (b) a DNA comprising the nucleotide sequence of SEQ ID NO: 1, wherein one or more nucleotides are replaced, deleted, inserted, and/or added. [6] The transgenic silkworm of [4], wherein the DNA encoding an RNA molecule exhibiting an RNAi effect is a DNA comprising the nucleotide sequence of SEQ ID NO: 2* [7] A method for producing a transgenic silkworm that shows resistance against nuclear polyhedrosis viruses, wherein the method comprises the steps of: (a) transferring into silkworm eggs a DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses; and (b) selecting a transgenic silkworm from among silkworms obtained from the eggs to which said DNA was transferred, [8] A method for making silkworms resistant to nuclear polyhedrosis viruses, wherein the method comprises the step of expressing in silkworm cells a DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses. [9] The method of [7] or [8], wherein the DNA encoding an RNA molecule is a DNA encoding an RNA molecule with a hahpin-type structure, [10] The method of [9]s wherein the DNA encoding an RNA molecule with a haiipin-type structure is a DNA in which a sense code DNA encoding a sense RNA of any one of the regions of an mRNA encoding a gene essential for proliferation of nuclear polyhedrosis viruses, and a DNA complementary to said sense code DNA are linked through a linker such that the sense code DNA and complementary DNA face each other. [11] The method of any one of [7] to [10], wherein the gene essential for proliferation of nuclear polyhedrosis viruses is the lefl gene. [12] A vector comprising a DNA that encodes an RNA molecule exhibiting an RNAi effect on a gene essential for proliferation of nuclear polyhedrosis viruses, wherein said DNA is inserted between inverted terminal repeats of a transposon and functionally linked to a promoter; said DNA comprising a sense code DNA that encodes a sense RNA of any one of the regions of an mRNA encoding said gene, and a complementary DNA thereof; wherein said sense code and complementary DNAs are linked through a linker so that they face each other. [13] The vector of [12j5 wherein the gene essential for proliferation of nuclear polyhedrosis viruses is the lefl gene. [14] The vector of [13], wherein the DNA encoding the sense RNA is the DNA of (a) or (b): (a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1; (b) a DNA comprising the nucleotide sequence of SEQ ID NO: 1, wherein one or more nucleotides are replaced, deleted, inserted, and/or added. [15] The vector of [13], wherein the DNA encoding the RNAmolecule exhibiting an RNAi effect is a DNA comprising the nucleotide sequence of SEQ ID NO: 2. [16] A kit comprising the vector of any one of [12] to [15] and a vector having a DNA that encodes a transposase. [17] An agent for making silkworms resistant to nuclear polyhedrosis viruses, wherein the agent comprises the vector of any one of [12] to [15], or the kit of [16]. BRIEF DESCRIPTION OF THE DRAWINGS Fig, 1 shows the structure of the vector used to produce the transgenic silkworms. P indicates the virus-derived SKI 8 promoter, lef indicates a portion of the lefl gene involved in viral replication (the orientation of the gene is indicated by arrows), Fbp(A) is a silkworm fibroin gene-derived poly A signal, and A3pGFP indicates the marker gene for differentiating the transgenic silkworms. A 96-nucleotide long linker exists between the sense DNA and antisense DNA of lefl. Fig. 2 shows the proliferation of viruses in transgenic silkworms carrying the virus-resistance gene. The amount of viruses in the blood was measured 72 hours and 96 hours after a set amount of the virus (105 viral polyhedron per individual) was fed to the transgenic silkworms (tg) and the control group. The amount of virus was determined by quantifying the viral DNA by real-time PCR> DETAILED DESCRIPTION OF THE INVENTION The present inventors were the first to successfully make silkworms resistant to NPV by inducing expression of a DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of NPV in silkworm cells. The present invention is based on this finding, and provides transgenic silkworms showing resistance against NPV, namely transgenic silkworms carrying an expressive DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of NPV. In the present invention, the meaning of the phrase 'transgenic silkworms carrying an expressive DNA encoding an RNAmolecule exhibiting an RNAi effect against a gene essential for proliferation of NPV" encompasses not only transgenic silkworms that constitutively express the DNA, but also includes transgenic silkworms that express the DNA under specific conditions (i.e.? inducible expression), The DNA of this invention that encodes an RN A molecule exhibiting an RNAi effect includes a DNA encoding an RNA molecule, a metabolite of which exhibits an RNAi effect (for example, a product made through cleavage on an RNA chain). The DNA of this invention that encodes an RNA molecule exhibiting an RNAi effect is preferably a DNA encoding an RNA molecule with a hairpin-type structure* Examples of the DNA encoding an RNA molecule with a hairpin-type structure include, but are not limited to, a DNA in which a sense (or antisense) code DNA encoding a sense (or antisense) RNA of any one of the regions of an mRNA encoding a gene essential for proliferation of NPV* and a DNA complementary to this DNA are linked through a linker such that they face each other. Herein, the term "face each other' means that two sequences are positioned in opposite orientation to each other. In other words, the DNA of this invention has a structure in which an inverted repeated sequence is formed by linking the DNA encoding the sense RNA through a linker. More specifically, when the DNA sequence (duplex) encoding a sense RNA is for example: AGTC (sense strand) • ' * • ?»» TCAG (antisense strand), the structure of the above-mentioned DNA sequence encoding the sense RNA that forms an inverted repeated sequence through a linker can be represented by: AGTC-LLL-GACT TCAG-LLL-CTGA (herein "L" denotes a discretionary nucleotide of the linker, and *:" denotes hydrogen bonds). The DNA of this invention preferably has a structure in which a sense code DNA that encodes a sense RNA of any one of the regions of a target gene mRNA, and a DNA complementary to this DNA are linked through a linker such that they face each other, as described above- The "complementary DNA" may be described more specifically as a DNA encoding an RNA antisense to "any one of the regions of the target gene mRNA" mentioned above. Therefore, in other words, the DNA of this invention may be described as having a structure in which an antisense code DNA encoding an RNA antisense to any one of the regions of a target gene mRNA and a DNA complementary to this DNA are linked in opposite directions through a linker. Since the DNA of this invention has a linker between lie inverted repeated sequences, the transcription product of the DNA of this invention also has a structure containing a linker between the inverted repeated sequences. An RNA molecule having such a structure ordinarily forms hydrogen bonds between the repeated sequences, and forms a hairpin structure. The length of the DNA constituting the linker of this invention is not particularly limited, so long as the adjacent repeated sequences can form hydrogen bonds. However, when introns are not included, it ordinarily ranges from a few nucleotides (e.g., 1 to 10 nucleotides) to approximately 100 nucleotides, more preferably several tens of nucleotides (e.g., 60,70, 80, or 90 nucleotides). The nucleotide sequence of the DNA constituting the linker is not particularly limited, and may be a discretionary sequence. Furthermore, so long as hydrogen bonds may be formed between the inverted repeated sequences as described above, a linker is not absolutely necessary. Accordingly, the DNA of this invention includes DNA in which the linker is absent. The length of the sense code DNA in the DNA of this invention, or a DNA that is complementary to this DNA, is ordinarily 1000 nucleotides or less, more preferably approximately 500 nucleotides. Furthermore, in the present invention, the duplex RNA portion in which RNAs are paired need not be completely paired, and, in fact, may contain unpaired sites. Unpaired sites can be established within a range that would not interfere with RNA formation. The DNA of this invention can be produced by those skilled in the art using conventional genetic engineering techniques. For example, it can be produced by performing PCR amplifications on 526 nucleotides from the 5 s end, and on 430 nucleotides from the 5 s end of the ORF sequence of lefl, respectively, and by linking them in opposite orientations. Examples of the gene essential for proliferation of NPV in this invention include the lefl gene, ie-1 gene, and pi 43 (DNAhelicase) gene. The nucleotide sequences of these genes are disclosed in the nucleotide sequences shown in Accession No: NC_001962, and are ORF6 (GENE ID: 1488636), ORF123 (GENE ID: 1488755), and ORF78 (GENE ID: 1724488) for the lefl gene, ie-1 gene, and pl43 gene, respectively (Sumiko Gomi, Kei Majima and Susumu Maeda, Journal of General Virology (1999), 80,1323-1337). It is a well known fact to those skilled in the art that viral DNAs can easily mutate. Therefore, the genes of this invention are not limited to the genes comprising the disclosed sequences, so long as the mutated gene is essential to proliferation of NPV. When the lefl gene is selected as the gene essential for NPV proliferation, examples of the sense code DNA in the DNA encoding the RNA molecule include a DNA comprising the nucleotide sequence of SEQ ID NO: 1, but are not limited thereto. DNA comprising the nucleotide sequence of SEQ ID NO: 1 wherein one or more nucleotides are replaced, deleted, inserted, and/or added may also be used. Mutations in the nucleotide sequence of SEQ ID NO: 1 which involve substitution, deletion, insertion, and/or addition of one or more nucleotides, include mutations that are not only caused naturally but also induced artificially. There is no limitation on the number of mutations and mutation sites of nucleic acid in the nucleotide sequence, so long as the nucleotide sequence can be used to express an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses. The number of mutations is typically no more than 10%3 preferably no more than. 5%, more preferably no more than 1%, of all nucleic acids. When the lefl gene is selected as the target gene, examples of the DNA encoding an RNA molecule with a hairpin-type structure include a DNA comprising the nucleotide sequence of SEQ ID NO: 2 (positions 1 to 430: lefl site sense sequence positions 431 to 526: linker sequence, and positions 527 to 956: antisense sequence), but are not limited thereto. A method for producing transgenic silkworms of this invention will be described using examples; however, the transgenic silkworms of this invention are not limited to those produced by the exemplary method described. For example, the transgenic silkworms of this invention can be produced by transferring a DNA encoding an RNA molecule exhibiting RNAi effect against a gene essential for NPV proliferation, into silkworm eggs, and then selecting the transgenic silkworms from silkworms that hatched from the eggs carrying the DNA. For example, transfer of DNA into silkworm eggs can be carried out by injecting piggyBac transposon as a vector into silkworm eggs in the early stage of development (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. and Couble, P., 2000, Nature Biotechnology 18, 81-84). For example, a vector containing a DNA between the inverted terminal repeats of a transposon (Handler, A.M., McCombs, S.D., Fraser, M J., Saul, S.H. (1998) Proc. Natl. Acad. Sci. U.S.A. 95(13):7520-5)„ in which this DNA has the DNA encoding an RNA molecule exhibiting an RNAi effect targeting a gene essential for proliferation of NPV, functionally linked downstream of a discretionary promoter, is transferred into silkworm eggs along with a vector (helper vector) carrying a DNA encoding a transposase In the present invention, the phrase "functionally linked" means that the promoter and the DNA are linked such that binding of a transcription factor to the promoter induces expression of the DNA positioned downstream of the promoter. Accordingly, a discretionary DNA sequence may be present between the promoter and the DNA as long as transcription of the DNA can take place* Examples of the promoters suitable for use in the present invention include, but are not limited to, promoters of genes that may be expressed in any tissue, such as the SKI 8 promoter, promoter of a heat shock protein gene, and promoters of cytoplasmic actin and tRNA gene, heterogeneous promoter system such as the GAL4AJAS system that utilizes the transcription regulatory system, and promoters of genes that are expressed specifically in the fat body and midgut. Furthermore, examples of the helper vector include, but are not limited to, pHA3PIG (Tamura, T.; Thibert, C.B Royer »C, Kanda, T,, Abraham, R, Kamba, M.„ Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G, Shirk, P., Fraser, M., Prudhomme, 1-C. and Couble, P., 2000, Nature Biotechnology 18, 81-84), but are not limited thereto. Examples of the transposon of this invention include piggyBac, but are not limited thereto* as mariner or minos may be used (Shimizu, K., Kamba, M.f Sonobe, R, Kanda, T.s Klinakis, A. Gs Savakis, C. and Tamura, T. (2000) Insect Mol. Biol, 9,277-281; Wang W, Swevers L, Iatrou K. (2000) Insect Mol Biol 9 (2): 145-55). Abaculoviral vector may also be used to produce transgenic silkworms of the present invention (Yamao, M,» N. Katayama, H. Nakazawa, M. Yamakawa, Y Hayashi et aL91999, Genes Dev, 13:511-516). Hereinafter, methods for transferring DNA into silkworm eggs will be described in detail; however, the methods of this invention for transferring DNA into silkworm eggs are not limited thereto. For example, DNA may be transferred directly into eggs using a tube for injecting DNA into silkworm eggs. In a preferred embodiment, a hole is made, physically or chemically, in advance in an eggshell, and the DNA is transferred through the hole. In such an embodiment, the tube for DNA injection can be inserted into the egg through the hole by keeping the insertion angle nearly perpendicular to the ventral surface of the egg. In the present invention, an example of the method for physically making a hole in the eggshell may involve the use of needle or microlaser. Preferably, a hole can be made in an eggshell by a method using a needle. The material from which the needle is made, the strength of the needle, and such are not particularly restricted, so long as the needle can make a hole in the silkworm eggshell. The needle for use in the present invention ordinarily refers to a rod-shaped needle having a sharp tip; however, it is not limited to this form. So long as it can make a hole in an eggshell, there are no particular limitations on the overall shape- For example, a pyramid-shaped object with a sharp tip, and a cone-shaped object with a sharp tip are also included in the meaning of the term "needle" of this invention. In this invention, a tungsten needle is preferably used. The thickness (diameter) of the needle of this invention is such that it can make a hole that allows the capillary described below to pass through, the appropriate thickness ordinarily being 2 to 20 fxm, or preferably 5 to 10 ywL On the other hand, examples of methods for chemically making a hole in an eggshell include the method of making a hole using chemicals including, but not limited to, hypochlorous acid. In the present invention, the position where the hole is made is not particularly limited so long as it is a position where the insertion angle can be nearly perpendicular to the ventral surface of the egg when a tube for DNA injection is inserted from the hole. The hole is preferably on the ventral surface or the side opposite to it, more preferably on the ventral surface, or even more preferably slightly towards the back end of the central portion of the ventral surface of the egg. In the present invention, the phrase "nearly perpendicular" means 70° to 120°, more preferably 80° to 90°. In this invention, Btthe position that will develop into germ cells in the future" is ordinarily the position close to the egg surface at the ventral side of the egg (ordinarily, the position that is 0.01 mm to 0.05 mm from the egg surface), and is preferably the position slightly toward the posterior end in the position close to the egg surface at the ventral center of the egg. The tube for injecting DNA of this invention is not particularly limited in terms of its material, strength, internal diameter, and such; however,, when a hole is made physically or chemically to the egg shell before inserting the tube for DNA injection, preferably its thickness (external diameter) allows passage through the opened hole. Examples of such tubes include, but are not limited to, a glass capillary. In a preferred embodiment of the method for DNA transfer of this invention, the steps of physically or chemically making a hole in the silkworm egg, inserting the tube for DNA injection into the egg through the hole so that the insertion angle is nearly perpendicular to the ventral surface of the egg, and injecting the DNA are performed using a manipulator that both equips a needle and a tube for DNA injection. Ordinarily, the present invention is preferably carried out using an apparatus comprising the manipulator as one of the components. Such an apparatus is composed of a dissecting microscope, an illuminator, a movable stage, a coarse manipulator fixed to the microscope with a metal fitting, a micromanipulator attached to this manipulator, and an injector that adjusts the air pressure for DNA injection, The pressure used for the injector is provided from a nitrogen cylinder, and a pressure switch can be turned on using a foots witch. Injection is performed on eggs immobilized onto a substrate such as a glass slide, and the position of the eggs is set using a movable stage. The glass capillary of the micromanipulator is operated by an operator connected through four tubes. The actual procedure involves setting the position of the tungsten needle relative to the egg using a coarse manipulator, and making a hole by moving the egg horizontally using the stage lever. This is followed by operating the lever of the micromanipulator operator to guide the tip of the glass capillary to the position of the hole, and inserting the capillary into the egg using the stage lever. In this case, the glass capillary must be inserted so that it is perpendicular to the ventral surface of the egg. The footswitch is toned on to inject the DNA? and the capillary is drawn out of the egg by operating the lever. The opened hole is closed using instant adhesive or such, and the eggs are protected in an incubator at constant temperature and constant humidity. The apparatus used for this invention is preferably the apparatus described in Japanese Patent No. 1654050, or an improved version of this apparatus. Furthermore, in an embodiment of the present invention, the silkworm eggs used for DNA transfer are preferably immobilized onto a substrate. Examples of substrates suitable for use in the present invention include a slide glass and plastic sheet, though the invention is not particularly limited thereto. In the embodiment of this invention, desirably, the eggs are immobilized after the orientations are properly arranged, so that the DNA can be injeeted precisely tn the pnsrrinn in fhe silkworm egg l.hal will develop into germ cells in the future. Furthermore, in this embodiment, the number of silkworm eggs immobilized onto the substrate is not particularly limited. When a plurality of silkworm eggs are used, the silkworm eggs are proforably waraiobilizod onto the aubotrate by orienting them euch that th© doreoventral directions are unidirectional* The immobilization of the silkworm eggs to a substrate according to this invention can be performed, for example by inducing oviposition on a commercially available paper (loose egg cards) pre-plastered with water-soluble glue, detaching the eggs by adding water to the paper, then placing the wet eggs as an array on a substrate, and then air-drying it. The eggs are preferably immobilized onto a slide glass by properly arranging the orientation of the eggs. Furthermore, immobilization of the eggs to the substrate can be accomplished by using a double-sided adhesive tape, adhesive agent, or such. Examples of the methods for investigating whether the DNA has been transferred to the silkworm eggs include the method of re-extracting the injected DNA from the eggs and measuring it (Nagaraju, J,, Kanda, TM Yukuhiro, K., Chavancy, G, Tamura, TM & Couble, P. (1996), Attempt of transgenesis of the silkworm (Bombyx mori L) by egg-injection of foreign DNA. Appl. Entomol. ZooL, 31, 589-598), or the method of observing the expression of the DNA as injected genes in the eggs (Tamura, T., Kanda, T., Takiya, S,9 Okano, K., & Maekawa, H. (1990). Transient expression of chimeric CAT genes injected into early embryos of the domesticated silkworm, Bombyx mori. Jpn. J. Genet., 65,401-410). In the methods for producing transgenic silkworms of this invention, the next step involves selecting transgenic silkworms from among the silkworms that hatched from the DNA-transferred eggs of this invention. For example, in this invention, transgenic silkworms can be selected using selection markers. For the selection markers of this invention, markers that are generally used by those skilled in the art may be used, including, but not limited to, fluorescent proteins such as CFP, GFP (for example, A3GFP), YFP, and DsRed. By using these markers, transgenic silkworms can be detected simply by observation through a fluorescence stereoscopic microscope. Furthermore, since the fluorescent colors differ, a plurality of markers can be used simultaneously. The present invention further provides vectors for use in the above-mentioned methods, and their kits. More specifically, this invention provides vectors comprising a DNAthat encodes an RNA molecule exhibiting an RNAi effect on a gene essential for proliferation of NPV. In a preferred embodiment, the DNA is inserted between the inverted terminal repeats of a transposon, is functionally linked to a promoter, and comprises a sense code DNAthat encodes a sense RNA of any one of the regions of the mRNA encoding the gene, and a DNA complementary to the DNAS linked through a linker such that they face each other. Examples of such vectors include, but are not limited to, a vector in which a DNA has been linked downstream of a promoter such as SKI 8 and inserted between the inverted terminal repeats of the transposon of a piggyBac-derived vector (Tamura, T., et al> 2000, Nature Biotechnology 18, 81-84), wherein the inserted DNA has a DNA encoding a sense (or antisense) code DNAthat encodes a sense (or antisense) RNA of any one of the regions of the mRNA encoding a gene essential to NPV proliferation, and a DNA complementary to this DNA linked through a linker such that they face each other. When the lefl gene is selected as the gene essential for NPV proliferation in this vector, a DNA comprising the previously mentioned sequence can be used as the sense code DNA and the DNA encoding an RNA molecule with a hairpin-type structure. The present invention also provides a kit comprising the above-mentioned vector, and a vector (helper vector) comprising a DNA encoding a transposase. Examples of the helper vector include, but are not limited to, pHA3PIG (Tamura, T., et al9 2000, Nature Biotechnology 18, 81-84). Furthermore, the present invention provides uses of the vectors and kits of this invention. More specifically, this invention provides uses as agents that make silkworms resistant to NPV, in which the agents comprise as the active ingredient the vector or the kit of this invention. The industrial utility of this invention is that it can provide transgenic silkworms showing resistance against NPV by producing transgenic silkworms carrying a gene which is a recombinant viral gene. Production of silkworms showing resistance against NPV will have an immense effect since this will enable avoidance of damages by NPV, which is the most serious problem at the work site of sericultural industry. This, in turn, will result in increased efficiency in the production of silkworm varieties, saving labor needed for farming, and production of high quality silk. Any patents, patent applications, and publications cited herein are incorporated by reference, EXAMPLES The present invention will be explained in detail below with reference to examples; however,, it is not to be construed as being limited thereto. In order to suppress viral growth, a virus-derived gene was recombined to produce a gene that makes mRNA with a hairpin structure. The structure of the produced gene and vector are shown in Fig. 1. The lefl gene is a gene that relates to replication of NPV DNA in silkworms, and is a gene that is essential for proliferation of the virus. A gene in which the transcription direction of this gene has been reversed was linked, and SKI 8 showing strong activity in cultured cells was used as its promoter. Furthermore, to distinguish transgenic silkworms carrying this gene, the A3GFP gene was used as a marker gene that enables expression of the fluorescent protein in the entire body, and allows easy discrimination of an individual carrying the gene under a fluorescence stereoscopic microscope. This vector DNA and a DNA (helper DNA) that expresses a factor having the effect of introducing the gene of interest into a genome were injected into silkworm eggs in the early stage of development. Larvae that hatched from the eggs were raised until they became adults* and then the second generation was obtained. Individuals whose entire bodies emitted fluorescence were identified by observation of these second generation larvae under a fluorescence microscope. Since these individuals appeared to be transgenic silkworms, they were continued to be raised and systematized. A fixed amount of the NPV polyhedron was fed to the systemized last instar transgenic silkworm, and proliferation of the virus in the blood was investigated 72 hours later and 96 hours later by real-time PCR. The results showed that in silkworms carrying the gene of Fig* ls proliferation of the virus was remarkably suppressed as shown in Fig. 2. The results described above showed that production of transgenic silkworms that carry a gene produced by recombining a viral gene can lead to production of virus-resistant silkworms. CLAIMS 1. A transgenic silkworm that shows resistance against nuclear polyhedrosis viruses, wherein the silkworm carries an expressive DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses. 2. The transgenic silkworm of claim 1,wherein the DNA encoding an RNA molecule is a DNA encoding an RNA molecule with a hairpin-type structure. 3. The transgenic silkworm of claim 2, wherein the DNA encoding an RNA molecule with a hairpin-type structure is a DNA in which a sense code DNA encoding a sense RNA of any one of the regions of an mRNA encoding a gene essential for proliferation of nuclear polyhedrosis viruses, and a DNA complementary to said sense code DNA are linked through a linker such that the sense code DNA and complementary DNA face each other. 4. The transgenic silkworm of any one of claims 1 to 3, wherein the gene essential for proliferation of nuclear polyhedrosis viruses is the lefl gene* 5. The transgenic silkworm of claim 4, wherein the DNA encoding a sense RNA is the DNA of (a)or(b): (a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1; (b) a DNA comprising the nucleotide sequence of SEQ ID NO: 1, wherein one or more nucleotides are replaced, deleted, inserted, and/or added. 6. The transgenic silkworm of claim 4, wherein the DNA encoding an RNA molecule exhibiting an RNAi effect is a DNA comprising the nucleotide sequence of SEQ ID NO: 2. 7. A method for producing a transgenic silkworm that shows resistance against nuclear polyhedrosis viruses, wherein the method comprises the steps of: (a) transferring into silkworm eggs a DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses; and (b) selecting a transgenic silkworm from among silkworms obtained from the eggs to which said DNA was transferred. 8. A method for making silkworms resistant to nuclear polyhedrosis viruses, wherein the method comprises the step of expressing in silkworm cells a DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses. 9. The method of claim 7 or 8, wherein the DNA encoding an RNA molecule is a DNA encoding an RNA molecule with a hairpin-type structure. 10. The method of claim 9, wherein the DNA encoding an RNA molecule with a hairpin-type structure is a DNA in which a sense code DNA encoding a sense RNA of any one of the regions of an mRNA encoding a gene essential for proliferation of nuclear polyhedrosis viruses, and a DNA complementary to said sense code DNA are linked through a linker such that the sense code DNA and complementary DNA face each other. 11. The method of any one of claims 7 to 10, wherein the gene essential for proliferation of nuclear polyhedrosis viruses is the lefl gene. 12. A vector comprising a DNA that encodes an RNA molecule exhibiting an RNAi effect on a gene essential for proliferation of nuclear polyhedrosis viruses, wherein said DNA is inserted between inverted terminal repeats of a transposon and functionally linked to a promoter; said DNA comprising a sense code DNA that encodes a sense RNA of any one of the regions of an mRNA encoding said gene, and a complementary DNA thereof; wherein said sense code and complementary DNAs are linked through a linker so that they face each other. 13. The vector of claim 12, wherein the gene essential for proliferation of nuclear polyhedrosis viruses is the lefl gene. 14. The vector of claim 13, wherein the DNA encoding the sense RNA is the DNA of (a) or (b): (a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1; (b) a DNA comprising the nucleotide sequence of SEQ ID NO; 1, wherein one or more nucleotides are replaced, deleted, inserted, and/or added, 15. The vector of claim 13, wherein the DNA encoding the RNAmolecule exhibiting an RNAi effect is a DNA comprising the nucleotide sequence of SEQ ID NO: 2. 16. A kit comprising the vector of any one of claims 12 to 15 and a vector having a DNA that encodes a transposase. 17. An agent for making silkworms resistant to nuclear polyhedrosis viruses, wherein the agent comprises the vector of any one of claims 12 to 15, or the kit of claim 16. Dated this 25 day of May 2005

Full Text

TRANSGENIC SILKWORMS THAT SHOW RESISTANCE AGAINST NUCLEAR POLYHEDROSIS VIRUSES, AND METHODS FOR PRODUCING THEM
FIELD OF THE INVENTION The present invention relates to transgenic silkworms that show resistance against nuclear polyhedrosis viruses. Furthermore, this invention relates to methods for producing transgenic silkworms that show resistance against nuclear polyhedrosis viruses.
BACKGROUND OF THE INVENTION
At the work site of sericultural industry, silkworms are seriously damaged by nuclear polyhedrosis viruses (NPV). To date, many researchers have worked on the production of NPV-resistant silkworms, but, to date, no one has yet succeeded. This is due to the fact that the gene for resistance against NPV is absent in all silkworms preserved in the world. To prevent damages from these viruses, thorough sterilization of the silkworm incubation areas and related facilities has been encouraged. However, for sericulture farms, such treatment is not only troublesome but also involves tremendous labor and resource costs, makes cocoons costly, and is one of the factors complicating farming itself.
NPV is a large DNA-type virus that carries at least 50 or more genes. It is known to proliferate and express these genes in host silkworm cells. A number of these genes are known to be essential for viral growth (Kool M, Ahrens CHS Goldbach RW, Rohnnann GF5 Vlak JM. (1994) Identification of genes involved in DNA replication of the Autographa califoraica baculovirus. Proc.Natl. Acad. SciUSA91(23): 11212-11216; LuA, Miller IX (1995) The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication. J.
Virol 69(2): 975-982).
Recently, new methods for producing transgenic silkworms, utilizing transposons or marker genes, have been developed (Tamura, T. (1999) Methods for producing transformed silkworms using transposons. Abstracts of the 7th Conference on Insect Functions, pi 0-22; Horn, C, B. Jaunich and E. A. Wimmer, (2000) Highly sensitive, fluorescent transformation marker for Drosophila transgenesis, Dev Genes Evol 210: 623-629; Horn, C.? and E. A, Wimmer, (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210: 630-637; Thomas, J. L., M. Da Rocha, A. Besse, B. Mauchamp and G Chavancy, 2002 3xP3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the embryonic stage onwards. Insect Biochem Mol Biol 32:247-253; Tamura, T., Thibert, C., Royer, C, Kanda, X, Abraham, E*s Kamba, M, Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G, Shirk, P., Fraser, M, Prudhomme, J.-C. and Couble, P. (2000) A piggyBac element-derived vector efficiently promotes germ-line transformation in the silkworm Bombyx mori L. Nature Biotechnology 18, 81-84; Berghammer,

I
A. J., M. Klingler and E. A. Wimmer, (1999) A universal marker for transgenic insects. Nature 402: 370-371; Tomita, M., Sato, T., Adachi,15 Munetsuna, H., Tamura, T., Kanda, T.f Yoshizato, K, (2001) Production of human collagen gene-incorporated transgenic silkworms. Abstracts of the 24th Annual Meeting of the Molecular Biology Society of Japan). The methods use DNA-type piggyBac transposon as the vector, and the green fluorescent protein (GFP) gene as the marker gene. Since these methods can efficiently produce recombinants, they may be used under various practical situations, such as production of useful proteins (Tomita, M.„ M. H., Sato T, Adachi T, Hino R, Hayashi M, Shimizu K, Nakamura N, Tamura T, Yoshizato a K.s (2003) Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nat BiotechnoL 21,52-56.) and new fibers, and development of varieties that are granted resistance against viruses. Accordingly, parts of such studies have been undertaken.
SUMMARY OF THE INVENTION
The present invention was made in view of the above-mentioned circumstances. An objective of the present invention is to provide transgenic silkworms showing resistance against NPV, and methods for producing them. More specifically, the objective is to provide NPV-resistant transgenic silkworms which carry an expressive DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for NPV proliferation, and methods for producing such silkworms.
The present inventors carried out extensive analyses to achieve the above-mentioned objective. First, the inventors examined whether NPV-resistant silkworms could be produced by utilizing transgenic silkworms. More specifically, a gene that forms an mRNA with a hairpin-type structure was artificially produced utilizing a virus-derived gene as the template, and NPV proliferation in transgenic silkworms carrying this gene was investigated. As a result, proliferation of viruses was found to be significantly suppressed.
More specifically, the present invention relates to transgenic silkworms showing resistance against NPV and methods for producing them. Specific objectives of the present invention, objects [1] to [17], are described below.
[1] A transgenic silkworm that shows resistance against nuclear polyhedrosis viruses* wherein the silkworm carries an expressive DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses. [2] The transgenic silkworm of [1], wherein the DNA encoding an RNA molecule is a DNA encoding an RNA molecule with a hairpin-type structure,
[3] The transgenic silkworm of [2], wherein the DNA encoding an RNA molecule with a hairpin-type structure is a DNA in which a sense code DNA encoding a sense RNA of any one of the regions of an mRNA encoding a gene essential for proliferation of nuclear polyhedrosis

viruses, and a DNA complementary to said sense code DNA are linked through a linker such that
the sense code DNA and complementary DNA face each other.
[4] The transgenic silkworm of any one of [1] to [3], wherein the gene essential for
proliferation of nuclear polyhedrosis viruses is the lefl gene.
[5] The transgenic silkworm of [4], wherein the DNA encoding a sense RNA is the DNA of (a)
or(b):
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1;
(b) a DNA comprising the nucleotide sequence of SEQ ID NO: 1, wherein one or more nucleotides are replaced, deleted, inserted, and/or added.
[6] The transgenic silkworm of [4], wherein the DNA encoding an RNA molecule exhibiting an RNAi effect is a DNA comprising the nucleotide sequence of SEQ ID NO: 2* [7] A method for producing a transgenic silkworm that shows resistance against nuclear polyhedrosis viruses, wherein the method comprises the steps of:
(a) transferring into silkworm eggs a DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses; and
(b) selecting a transgenic silkworm from among silkworms obtained from the eggs to which said DNA was transferred,
[8] A method for making silkworms resistant to nuclear polyhedrosis viruses, wherein the
method comprises the step of expressing in silkworm cells a DNA encoding an RNA molecule
exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis
viruses.
[9] The method of [7] or [8], wherein the DNA encoding an RNA molecule is a DNA encoding
an RNA molecule with a hahpin-type structure,
[10] The method of [9]s wherein the DNA encoding an RNA molecule with a haiipin-type
structure is a DNA in which a sense code DNA encoding a sense RNA of any one of the regions
of an mRNA encoding a gene essential for proliferation of nuclear polyhedrosis viruses, and a
DNA complementary to said sense code DNA are linked through a linker such that the sense
code DNA and complementary DNA face each other.
[11] The method of any one of [7] to [10], wherein the gene essential for proliferation of
nuclear polyhedrosis viruses is the lefl gene.
[12] A vector comprising a DNA that encodes an RNA molecule exhibiting an RNAi effect on
a gene essential for proliferation of nuclear polyhedrosis viruses, wherein said DNA is inserted
between inverted terminal repeats of a transposon and functionally linked to a promoter; said
DNA comprising a sense code DNA that encodes a sense RNA of any one of the regions of an
mRNA encoding said gene, and a complementary DNA thereof; wherein said sense code and
complementary DNAs are linked through a linker so that they face each other.

[13] The vector of [12j5 wherein the gene essential for proliferation of nuclear polyhedrosis
viruses is the lefl gene.
[14] The vector of [13], wherein the DNA encoding the sense RNA is the DNA of (a) or (b):
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1;
(b) a DNA comprising the nucleotide sequence of SEQ ID NO: 1, wherein one or more nucleotides are replaced, deleted, inserted, and/or added.
[15] The vector of [13], wherein the DNA encoding the RNAmolecule exhibiting an RNAi
effect is a DNA comprising the nucleotide sequence of SEQ ID NO: 2.
[16] A kit comprising the vector of any one of [12] to [15] and a vector having a DNA that
encodes a transposase.
[17] An agent for making silkworms resistant to nuclear polyhedrosis viruses, wherein the
agent comprises the vector of any one of [12] to [15], or the kit of [16].
BRIEF DESCRIPTION OF THE DRAWINGS Fig, 1 shows the structure of the vector used to produce the transgenic silkworms. P indicates the virus-derived SKI 8 promoter, lef indicates a portion of the lefl gene involved in viral replication (the orientation of the gene is indicated by arrows), Fbp(A) is a silkworm fibroin gene-derived poly A signal, and A3pGFP indicates the marker gene for differentiating the transgenic silkworms. A 96-nucleotide long linker exists between the sense DNA and antisense DNA of lefl.
Fig. 2 shows the proliferation of viruses in transgenic silkworms carrying the virus-resistance gene. The amount of viruses in the blood was measured 72 hours and 96 hours after a set amount of the virus (105 viral polyhedron per individual) was fed to the transgenic silkworms (tg) and the control group. The amount of virus was determined by quantifying the viral DNA by real-time PCR>
DETAILED DESCRIPTION OF THE INVENTION The present inventors were the first to successfully make silkworms resistant to NPV by
inducing expression of a DNA encoding an RNA molecule exhibiting an RNAi effect against a
gene essential for proliferation of NPV in silkworm cells.
The present invention is based on this finding, and provides transgenic silkworms
showing resistance against NPV, namely transgenic silkworms carrying an expressive DNA
encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation
of NPV.
In the present invention, the meaning of the phrase 'transgenic silkworms carrying an expressive DNA encoding an RNAmolecule exhibiting an RNAi effect against a gene essential

for proliferation of NPV" encompasses not only transgenic silkworms that constitutively express the DNA, but also includes transgenic silkworms that express the DNA under specific conditions (i.e.? inducible expression),
The DNA of this invention that encodes an RN A molecule exhibiting an RNAi effect includes a DNA encoding an RNA molecule, a metabolite of which exhibits an RNAi effect (for example, a product made through cleavage on an RNA chain).
The DNA of this invention that encodes an RNA molecule exhibiting an RNAi effect is preferably a DNA encoding an RNA molecule with a hairpin-type structure* Examples of the DNA encoding an RNA molecule with a hairpin-type structure include, but are not limited to, a DNA in which a sense (or antisense) code DNA encoding a sense (or antisense) RNA of any one of the regions of an mRNA encoding a gene essential for proliferation of NPV* and a DNA complementary to this DNA are linked through a linker such that they face each other.
Herein, the term "face each other*' means that two sequences are positioned in opposite orientation to each other. In other words, the DNA of this invention has a structure in which an inverted repeated sequence is formed by linking the DNA encoding the sense RNA through a linker. More specifically, when the DNA sequence (duplex) encoding a sense RNA is for example:
AGTC (sense strand)
• * ' *
• ?»»
TCAG (antisense strand),
the structure of the above-mentioned DNA sequence encoding the sense RNA that forms an
inverted repeated sequence through a linker can be represented by:
AGTC-LLL-GACT
TCAG-LLL-CTGA
(herein "L" denotes a discretionary nucleotide of the linker, and **:" denotes hydrogen bonds).
The DNA of this invention preferably has a structure in which a sense code DNA that encodes a sense RNA of any one of the regions of a target gene mRNA, and a DNA complementary to this DNA are linked through a linker such that they face each other, as described above- The "complementary DNA" may be described more specifically as a DNA encoding an RNA antisense to "any one of the regions of the target gene mRNA" mentioned above. Therefore, in other words, the DNA of this invention may be described as having a structure in which an antisense code DNA encoding an RNA antisense to any one of the regions of a target gene mRNA and a DNA complementary to this DNA are linked in opposite directions through a linker.
Since the DNA of this invention has a linker between lie inverted repeated sequences,

the transcription product of the DNA of this invention also has a structure containing a linker between the inverted repeated sequences. An RNA molecule having such a structure ordinarily forms hydrogen bonds between the repeated sequences, and forms a hairpin structure.
The length of the DNA constituting the linker of this invention is not particularly limited, so long as the adjacent repeated sequences can form hydrogen bonds. However, when introns are not included, it ordinarily ranges from a few nucleotides (e.g., 1 to 10 nucleotides) to approximately 100 nucleotides, more preferably several tens of nucleotides (e.g., 60,70, 80, or 90 nucleotides).
The nucleotide sequence of the DNA constituting the linker is not particularly limited, and may be a discretionary sequence. Furthermore, so long as hydrogen bonds may be formed between the inverted repeated sequences as described above, a linker is not absolutely necessary. Accordingly, the DNA of this invention includes DNA in which the linker is absent.
The length of the sense code DNA in the DNA of this invention, or a DNA that is complementary to this DNA, is ordinarily 1000 nucleotides or less, more preferably approximately 500 nucleotides.
Furthermore, in the present invention, the duplex RNA portion in which RNAs are paired need not be completely paired, and, in fact, may contain unpaired sites. Unpaired sites can be established within a range that would not interfere with RNA formation.
The DNA of this invention can be produced by those skilled in the art using conventional genetic engineering techniques. For example, it can be produced by performing PCR amplifications on 526 nucleotides from the 5 s end, and on 430 nucleotides from the 5 s end of the ORF sequence of lefl, respectively, and by linking them in opposite orientations.
Examples of the gene essential for proliferation of NPV in this invention include the lefl gene, ie-1 gene, and pi 43 (DNAhelicase) gene. The nucleotide sequences of these genes are disclosed in the nucleotide sequences shown in Accession No: NC_001962, and are ORF6 (GENE ID: 1488636), ORF123 (GENE ID: 1488755), and ORF78 (GENE ID: 1724488) for the lefl gene, ie-1 gene, and pl43 gene, respectively (Sumiko Gomi, Kei Majima and Susumu Maeda, Journal of General Virology (1999), 80,1323-1337).
It is a well known fact to those skilled in the art that viral DNAs can easily mutate. Therefore, the genes of this invention are not limited to the genes comprising the disclosed sequences, so long as the mutated gene is essential to proliferation of NPV.
When the lefl gene is selected as the gene essential for NPV proliferation, examples of the sense code DNA in the DNA encoding the RNA molecule include a DNA comprising the nucleotide sequence of SEQ ID NO: 1, but are not limited thereto. DNA comprising the nucleotide sequence of SEQ ID NO: 1 wherein one or more nucleotides are replaced, deleted, inserted, and/or added may also be used.

Mutations in the nucleotide sequence of SEQ ID NO: 1 which involve substitution, deletion, insertion, and/or addition of one or more nucleotides, include mutations that are not only caused naturally but also induced artificially. There is no limitation on the number of mutations and mutation sites of nucleic acid in the nucleotide sequence, so long as the nucleotide sequence can be used to express an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses. The number of mutations is typically no more than 10%3 preferably no more than. 5%, more preferably no more than 1%, of all nucleic acids.
When the lefl gene is selected as the target gene, examples of the DNA encoding an RNA molecule with a hairpin-type structure include a DNA comprising the nucleotide sequence of SEQ ID NO: 2 (positions 1 to 430: lefl site sense sequence* positions 431 to 526: linker sequence, and positions 527 to 956: antisense sequence), but are not limited thereto.
A method for producing transgenic silkworms of this invention will be described using examples; however, the transgenic silkworms of this invention are not limited to those produced by the exemplary method described.
For example, the transgenic silkworms of this invention can be produced by transferring a DNA encoding an RNA molecule exhibiting RNAi effect against a gene essential for NPV proliferation, into silkworm eggs, and then selecting the transgenic silkworms from silkworms that hatched from the eggs carrying the DNA.
For example, transfer of DNA into silkworm eggs can be carried out by injecting piggyBac transposon as a vector into silkworm eggs in the early stage of development (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. and Couble, P., 2000, Nature Biotechnology 18, 81-84).
For example, a vector containing a DNA between the inverted terminal repeats of a transposon (Handler, A.M., McCombs, S.D., Fraser, M J., Saul, S.H. (1998) Proc. Natl. Acad. Sci. U.S.A. 95(13):7520-5)„ in which this DNA has the DNA encoding an RNA molecule exhibiting an RNAi effect targeting a gene essential for proliferation of NPV, functionally linked downstream of a discretionary promoter, is transferred into silkworm eggs along with a vector (helper vector) carrying a DNA encoding a transposase*
In the present invention, the phrase "functionally linked" means that the promoter and the DNA are linked such that binding of a transcription factor to the promoter induces expression of the DNA positioned downstream of the promoter. Accordingly, a discretionary DNA sequence may be present between the promoter and the DNA as long as transcription of the DNA can take place*
Examples of the promoters suitable for use in the present invention include, but are not

limited to, promoters of genes that may be expressed in any tissue, such as the SKI 8 promoter, promoter of a heat shock protein gene, and promoters of cytoplasmic actin and tRNA gene, heterogeneous promoter system such as the GAL4AJAS system that utilizes the transcription regulatory system, and promoters of genes that are expressed specifically in the fat body and midgut.
Furthermore, examples of the helper vector include, but are not limited to, pHA3PIG (Tamura, T.; Thibert, C.B Royer »C, Kanda, T,, Abraham, R, Kamba, M.„ Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G, Shirk, P., Fraser, M., Prudhomme, 1-C. and Couble, P., 2000, Nature Biotechnology 18, 81-84), but are not limited thereto.
Examples of the transposon of this invention include piggyBac, but are not limited thereto* as mariner or minos may be used (Shimizu, K., Kamba, M.f Sonobe, R, Kanda, T.s Klinakis, A. Gs Savakis, C. and Tamura, T. (2000) Insect Mol. Biol, 9,277-281; Wang W, Swevers L, Iatrou K. (2000) Insect Mol Biol 9 (2): 145-55).
Abaculoviral vector may also be used to produce transgenic silkworms of the present invention (Yamao, M,» N. Katayama, H. Nakazawa, M. Yamakawa, Y Hayashi et aL91999, Genes Dev, 13:511-516).
Hereinafter, methods for transferring DNA into silkworm eggs will be described in detail; however, the methods of this invention for transferring DNA into silkworm eggs are not limited thereto. For example, DNA may be transferred directly into eggs using a tube for injecting DNA into silkworm eggs. In a preferred embodiment, a hole is made, physically or chemically, in advance in an eggshell, and the DNA is transferred through the hole. In such an embodiment, the tube for DNA injection can be inserted into the egg through the hole by keeping the insertion angle nearly perpendicular to the ventral surface of the egg.
In the present invention, an example of the method for physically making a hole in the eggshell may involve the use of needle or microlaser. Preferably, a hole can be made in an eggshell by a method using a needle. The material from which the needle is made, the strength of the needle, and such are not particularly restricted, so long as the needle can make a hole in the silkworm eggshell. The needle for use in the present invention ordinarily refers to a rod-shaped needle having a sharp tip; however, it is not limited to this form. So long as it can make a hole in an eggshell, there are no particular limitations on the overall shape- For example, a pyramid-shaped object with a sharp tip, and a cone-shaped object with a sharp tip are also included in the meaning of the term "needle" of this invention. In this invention, a tungsten needle is preferably used. The thickness (diameter) of the needle of this invention is such that it can make a hole that allows the capillary described below to pass through, the appropriate thickness ordinarily being 2 to 20 fxm, or preferably 5 to 10 ywL On the other hand, examples of methods for chemically making a hole in an eggshell include the method of making

a hole using chemicals including, but not limited to, hypochlorous acid.
In the present invention, the position where the hole is made is not particularly limited so long as it is a position where the insertion angle can be nearly perpendicular to the ventral surface of the egg when a tube for DNA injection is inserted from the hole. The hole is preferably on the ventral surface or the side opposite to it, more preferably on the ventral surface, or even more preferably slightly towards the back end of the central portion of the ventral surface of the egg.
In the present invention, the phrase "nearly perpendicular" means 70° to 120°, more preferably 80° to 90°. In this invention, Btthe position that will develop into germ cells in the future" is ordinarily the position close to the egg surface at the ventral side of the egg (ordinarily, the position that is 0.01 mm to 0.05 mm from the egg surface), and is preferably the position slightly toward the posterior end in the position close to the egg surface at the ventral center of the egg.
The tube for injecting DNA of this invention is not particularly limited in terms of its material, strength, internal diameter, and such; however,, when a hole is made physically or chemically to the egg shell before inserting the tube for DNA injection, preferably its thickness (external diameter) allows passage through the opened hole. Examples of such tubes include, but are not limited to, a glass capillary.
In a preferred embodiment of the method for DNA transfer of this invention, the steps of physically or chemically making a hole in the silkworm egg, inserting the tube for DNA injection into the egg through the hole so that the insertion angle is nearly perpendicular to the ventral surface of the egg, and injecting the DNA are performed using a manipulator that both equips a needle and a tube for DNA injection. Ordinarily, the present invention is preferably carried out using an apparatus comprising the manipulator as one of the components.
Such an apparatus is composed of a dissecting microscope, an illuminator, a movable stage, a coarse manipulator fixed to the microscope with a metal fitting, a micromanipulator attached to this manipulator, and an injector that adjusts the air pressure for DNA injection, The pressure used for the injector is provided from a nitrogen cylinder, and a pressure switch can be turned on using a foots witch. Injection is performed on eggs immobilized onto a substrate such as a glass slide, and the position of the eggs is set using a movable stage. The glass capillary of the micromanipulator is operated by an operator connected through four tubes. The actual procedure involves setting the position of the tungsten needle relative to the egg using a coarse manipulator, and making a hole by moving the egg horizontally using the stage lever. This is followed by operating the lever of the micromanipulator operator to guide the tip of the glass capillary to the position of the hole, and inserting the capillary into the egg using the stage lever. In this case, the glass capillary must be inserted so that it is perpendicular to the ventral

surface of the egg. The footswitch is toned on to inject the DNA? and the capillary is drawn out of the egg by operating the lever. The opened hole is closed using instant adhesive or such, and the eggs are protected in an incubator at constant temperature and constant humidity. The apparatus used for this invention is preferably the apparatus described in Japanese Patent No. 1654050, or an improved version of this apparatus.
Furthermore, in an embodiment of the present invention, the silkworm eggs used for DNA transfer are preferably immobilized onto a substrate. Examples of substrates suitable for use in the present invention include a slide glass and plastic sheet, though the invention is not particularly limited thereto. In the embodiment of this invention, desirably, the eggs are immobilized after the orientations are properly arranged, so that the DNA can be injeeted
precisely tn the pnsrrinn in fhe silkworm egg l.hal will develop into germ cells in the future.
Furthermore, in this embodiment, the number of silkworm eggs immobilized onto the substrate is not particularly limited. When a plurality of silkworm eggs are used, the silkworm eggs are
proforably waraiobilizod onto the aubotrate by orienting them euch that th© doreoventral directions
are unidirectional* The immobilization of the silkworm eggs to a substrate according to this invention can be performed, for example by inducing oviposition on a commercially available paper (loose egg cards) pre-plastered with water-soluble glue, detaching the eggs by adding water to the paper, then placing the wet eggs as an array on a substrate, and then air-drying it. The eggs are preferably immobilized onto a slide glass by properly arranging the orientation of the eggs. Furthermore, immobilization of the eggs to the substrate can be accomplished by using a double-sided adhesive tape, adhesive agent, or such.
Examples of the methods for investigating whether the DNA has been transferred to the silkworm eggs include the method of re-extracting the injected DNA from the eggs and measuring it (Nagaraju, J,, Kanda, TM Yukuhiro, K., Chavancy, G, Tamura, TM & Couble, P. (1996), Attempt of transgenesis of the silkworm (Bombyx mori L) by egg-injection of foreign DNA. Appl. Entomol. ZooL, 31, 589-598), or the method of observing the expression of the DNA as injected genes in the eggs (Tamura, T., Kanda, T., Takiya, S,9 Okano, K., & Maekawa, H. (1990). Transient expression of chimeric CAT genes injected into early embryos of the domesticated silkworm, Bombyx mori. Jpn. J. Genet., 65,401-410).
In the methods for producing transgenic silkworms of this invention, the next step involves selecting transgenic silkworms from among the silkworms that hatched from the DNA-transferred eggs of this invention. For example, in this invention, transgenic silkworms can be selected using selection markers. For the selection markers of this invention, markers that are generally used by those skilled in the art may be used, including, but not limited to, fluorescent proteins such as CFP, GFP (for example, A3GFP), YFP, and DsRed. By using these markers, transgenic silkworms can be detected simply by observation through a fluorescence

stereoscopic microscope. Furthermore, since the fluorescent colors differ, a plurality of markers can be used simultaneously.
The present invention further provides vectors for use in the above-mentioned methods, and their kits. More specifically, this invention provides vectors comprising a DNAthat encodes an RNA molecule exhibiting an RNAi effect on a gene essential for proliferation of NPV. In a preferred embodiment, the DNA is inserted between the inverted terminal repeats of a transposon, is functionally linked to a promoter, and comprises a sense code DNAthat encodes a sense RNA of any one of the regions of the mRNA encoding the gene, and a DNA complementary to the DNAS linked through a linker such that they face each other. Examples of such vectors include, but are not limited to, a vector in which a DNA has been linked downstream of a promoter such as SKI 8 and inserted between the inverted terminal repeats of the transposon of a piggyBac-derived vector (Tamura, T., et al> 2000, Nature Biotechnology 18, 81-84), wherein the inserted DNA has a DNA encoding a sense (or antisense) code DNAthat encodes a sense (or antisense) RNA of any one of the regions of the mRNA encoding a gene essential to NPV proliferation, and a DNA complementary to this DNA linked through a linker such that they face each other. When the lefl gene is selected as the gene essential for NPV proliferation in this vector, a DNA comprising the previously mentioned sequence can be used as the sense code DNA and the DNA encoding an RNA molecule with a hairpin-type structure. The present invention also provides a kit comprising the above-mentioned vector, and a vector (helper vector) comprising a DNA encoding a transposase. Examples of the helper vector include, but are not limited to, pHA3PIG (Tamura, T., et al9 2000, Nature Biotechnology 18, 81-84). Furthermore, the present invention provides uses of the vectors and kits of this invention. More specifically, this invention provides uses as agents that make silkworms resistant to NPV, in which the agents comprise as the active ingredient the vector or the kit of this invention.
The industrial utility of this invention is that it can provide transgenic silkworms showing resistance against NPV by producing transgenic silkworms carrying a gene which is a recombinant viral gene.
Production of silkworms showing resistance against NPV will have an immense effect since this will enable avoidance of damages by NPV, which is the most serious problem at the work site of sericultural industry. This, in turn, will result in increased efficiency in the production of silkworm varieties, saving labor needed for farming, and production of high quality silk.
Any patents, patent applications, and publications cited herein are incorporated by reference,

EXAMPLES
The present invention will be explained in detail below with reference to examples; however,, it is not to be construed as being limited thereto.
In order to suppress viral growth, a virus-derived gene was recombined to produce a gene that makes mRNA with a hairpin structure. The structure of the produced gene and vector are shown in Fig. 1.
The lefl gene is a gene that relates to replication of NPV DNA in silkworms, and is a gene that is essential for proliferation of the virus. A gene in which the transcription direction of this gene has been reversed was linked, and SKI 8 showing strong activity in cultured cells was used as its promoter. Furthermore, to distinguish transgenic silkworms carrying this gene, the A3GFP gene was used as a marker gene that enables expression of the fluorescent protein in the entire body, and allows easy discrimination of an individual carrying the gene under a fluorescence stereoscopic microscope.
This vector DNA and a DNA (helper DNA) that expresses a factor having the effect of introducing the gene of interest into a genome were injected into silkworm eggs in the early stage of development. Larvae that hatched from the eggs were raised until they became adults* and then the second generation was obtained. Individuals whose entire bodies emitted fluorescence were identified by observation of these second generation larvae under a fluorescence microscope. Since these individuals appeared to be transgenic silkworms, they were continued to be raised and systematized. A fixed amount of the NPV polyhedron was fed to the systemized last instar transgenic silkworm, and proliferation of the virus in the blood was investigated 72 hours later and 96 hours later by real-time PCR. The results showed that in silkworms carrying the gene of Fig* ls proliferation of the virus was remarkably suppressed as shown in Fig. 2.
The results described above showed that production of transgenic silkworms that carry a gene produced by recombining a viral gene can lead to production of virus-resistant silkworms.

CLAIMS
1. A transgenic silkworm that shows resistance against nuclear polyhedrosis viruses, wherein the silkworm carries an expressive DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses.
2. The transgenic silkworm of claim 1,wherein the DNA encoding an RNA molecule is a DNA encoding an RNA molecule with a hairpin-type structure.
3. The transgenic silkworm of claim 2, wherein the DNA encoding an RNA molecule with a hairpin-type structure is a DNA in which a sense code DNA encoding a sense RNA of any one of the regions of an mRNA encoding a gene essential for proliferation of nuclear polyhedrosis viruses, and a DNA complementary to said sense code DNA are linked through a linker such that the sense code DNA and complementary DNA face each other.
4. The transgenic silkworm of any one of claims 1 to 3, wherein the gene essential for proliferation of nuclear polyhedrosis viruses is the lefl gene*
5. The transgenic silkworm of claim 4, wherein the DNA encoding a sense RNA is the DNA of (a)or(b):

(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1;
(b) a DNA comprising the nucleotide sequence of SEQ ID NO: 1, wherein one or more nucleotides are replaced, deleted, inserted, and/or added.

6. The transgenic silkworm of claim 4, wherein the DNA encoding an RNA molecule exhibiting an RNAi effect is a DNA comprising the nucleotide sequence of SEQ ID NO: 2.
7. A method for producing a transgenic silkworm that shows resistance against nuclear polyhedrosis viruses, wherein the method comprises the steps of:

(a) transferring into silkworm eggs a DNA encoding an RNA molecule exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses; and
(b) selecting a transgenic silkworm from among silkworms obtained from the eggs to which said DNA was transferred.
8. A method for making silkworms resistant to nuclear polyhedrosis viruses, wherein the
method comprises the step of expressing in silkworm cells a DNA encoding an RNA molecule

exhibiting an RNAi effect against a gene essential for proliferation of nuclear polyhedrosis viruses.
9. The method of claim 7 or 8, wherein the DNA encoding an RNA molecule is a DNA
encoding an RNA molecule with a hairpin-type structure.
10. The method of claim 9, wherein the DNA encoding an RNA molecule with a hairpin-type structure is a DNA in which a sense code DNA encoding a sense RNA of any one of the regions of an mRNA encoding a gene essential for proliferation of nuclear polyhedrosis viruses, and a DNA complementary to said sense code DNA are linked through a linker such that the sense code DNA and complementary DNA face each other.
11. The method of any one of claims 7 to 10, wherein the gene essential for proliferation of nuclear polyhedrosis viruses is the lefl gene.
12. A vector comprising a DNA that encodes an RNA molecule exhibiting an RNAi effect on a gene essential for proliferation of nuclear polyhedrosis viruses, wherein said DNA is inserted between inverted terminal repeats of a transposon and functionally linked to a promoter; said DNA comprising a sense code DNA that encodes a sense RNA of any one of the regions of an mRNA encoding said gene, and a complementary DNA thereof; wherein said sense code and complementary DNAs are linked through a linker so that they face each other.
13. The vector of claim 12, wherein the gene essential for proliferation of nuclear polyhedrosis viruses is the lefl gene.
14. The vector of claim 13, wherein the DNA encoding the sense RNA is the DNA of (a) or
(b):
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1;
(b) a DNA comprising the nucleotide sequence of SEQ ID NO; 1, wherein one or more nucleotides are replaced, deleted, inserted, and/or added,

15. The vector of claim 13, wherein the DNA encoding the RNAmolecule exhibiting an RNAi effect is a DNA comprising the nucleotide sequence of SEQ ID NO: 2.
16. A kit comprising the vector of any one of claims 12 to 15 and a vector having a DNA that encodes a transposase.

17. An agent for making silkworms resistant to nuclear polyhedrosis viruses, wherein the agent comprises the vector of any one of claims 12 to 15, or the kit of claim 16.
Dated this 25 day of May 2005

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Patent Number

241443

Indian Patent Application Number

633/CHE/2005

PG Journal Number

28/2010

Publication Date

09-Jul-2010

Grant Date

02-Jul-2010

Date of Filing

25-May-2005

Name of Patentee

NATIONAL INSTITUTE OF AGROBIOLOGICAL SCIENCES

Applicant Address

1-2, KANNONDAI 2-CHOME, TSUKUBA-SHI, IBARAKI 305-8602, JAPAN

Inventors:

#	Inventor's Name	Inventor's Address
1	TODHIKI TAMURA	C/O NATIONAL INSTITUTE OF AGROBIOLOGICAL SCIENCES, 1-2, KANNONDAI 2-CHOME, TSUKUBA-SHI, IBARAKI 305-8602, JAPAN
2	TOSHIO KANDA	C/O NATIONAL INSTITUTE OF AGROBIOLOGICAL SCIENCES, 1-2, KANNONDAI 2-CHOME, TSUKUBA-SHI, IBARAKI 305-8602, JAPAN
3	HISANORI BANDO	6-2-7, SHINKOTONIHACHIJO, KITA-KU, SAPPORO-SHI, HOKKAIDO 001-0908, JAPAN
4	HIDEO MATSUI	2-8-7, IWAGAMI-MACHI, MAEBASHI-SHI, GUNMA 371-0055, JAPAN
5	NUBUO KUWABARA	33-9, TSURUGAYAMA-MACHI, MAEBASHI-SHI, GUNMA 379-2108, JAPAN
6	KATSURA, KOJIMA	C/O NATIONAL INSTITUTE OF AGROBIOLOGICAL SCIENCES, 1-2, KANNONDAI 2-CHOME, TSUKUBA-SHI, IBARAKI 305-8602, JAPAN
7	JUN-ICHI MACHIDA	465, KAMINAMURO, KITATACHIBANA-MURA, SETA-GUN, GUNMA 377-0055, JAPAN

PCT International Classification Number

C12N 15/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	2004-159121	2004-05-28	Japan