Method for producing genetically modified cloned silkworms

Abstract

To develop and provide a method for making genetically modified clonal silkworms through a small number of steps with a high hatching rate of eggs after the gene transfer operation.SOLUTION: According to a method, non-dormant silkworms and dormant silkworms are crossbred, the resultant F1 fertilized eggs are subjected to gene transfer operation, unfertilized eggs are taken from a hatched F1 female individual, and genetically modified clonal silkworms are created.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a genetically modified cloned silkworm. [Background technology]

[0002] Genetic engineering of living organisms is essential for analyzing gene functions and producing useful proteins. Conventionally, E. coli and yeast have been used as host organisms for genetic recombination. These hosts have the advantage of being easy to cultivate and capable of mass-producing in a short period of time. , and the production of useful proteins that are raw materials for medical diagnostic reagents, cosmetics, veterinary drugs, and pharmaceuticals. In recent years, genetic engineering techniques have been established for silkworms, and proteins It has been attracting attention as a suitable host organism for mass production of bacteria.

[0003] The silkworm (Bombyx mori) is an insect that has long been used industrially to produce silk. Silkworms can produce a large amount of silk thread in a short period of time to make cocoons. This is due to the high protein production capacity of the plant. This ability of silkworms can now be used to mass-produce useful proteins other than silk. It is.

[0004] When silkworms are used as hosts for mass production of proteins, the following are produced: In other words, the technology to produce genetically modified silkworms (transgenic silkworms) is essential. Generally, to produce genetically modified silkworms, the desired gene is injected into eggs using a transposon. The microinjection method is used to inject silkworm eggs (Non-Patent Document 1). Fertilization occurs within two hours after birth, and then a bare nucleus without a cell membrane, called a syncytium, forms. The cells divide repeatedly in a short period of time and move to the surface of the egg. Specifically, within 2 to 8 hours, preferably 3 to 6 hours after laying, the eggs are transfected with target genes at both ends. Plasmids containing inverted terminal repeats of transposons and those with the function of mobilizing transposons By injecting a plasmid or messenger RNA that can produce transposase The target gene to be introduced is incorporated into the nucleus by the inverted terminal repeat sequence of the transposon, It can be inserted into the genome (Non-Patent Document 2).

[0005] However, in the transgenic silkworms obtained by the above method, the introduced gene of interest is Even if they are the same, the genome composition of each transformant is different. The lineage is maintained as a genetically heterogeneous population through hybridization, and This is because the insertion of the target gene by the poson occurs at a random position on the genome.

[0006] Silkworms are generally stored in the egg state, but the maximum storage period is one year. To maintain the lineage, they must be bred every year to produce eggs for the next generation. When crossbreeding is repeated over a long period of time, it becomes difficult to ensure genetic stability even within the same strain. Therefore, in conventional techniques, the expression level and quality of the target gene vary depending on the lineage of the transformant. or differs from individual to individual, resulting in inconsistent quality and production of the protein produced. This is because raw materials for pharmaceuticals, which require high quality, are genetically modified. This is a particularly big problem when manufacturing in mass production systems using ECO. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Tamura T. et al., 2000, Nature Biotechnology, 18, 81-84 [Non-patent document 2] Tamura, T. (2007) Development and Use of Methods for Producing Transgenic Silkworms (Series: 21st Century Agriculture: Genetic Engineering Research of Animals and Microorganisms, edited by the Agricultural Society of Japan), pp. 57-76. Yokendo, Tokyo. Summary of the Invention [Problem to be solved by the invention]

[0008] One solution to the above problem is to clone silkworms. They have the same genome composition and are a genetically homogeneous population. If cloning is possible, all cloned individuals will in principle be of the same quality and contain the same amount of protein. Therefore, it is possible to mass-produce proteins with stable quality and production volume. It becomes possible to do this.

[0009] Silkworms have a phenomenon called parthenogenesis, and cloned silkworms can be created by utilizing this phenomenon. Parthenogenesis in silkworms is induced by exposing unfertilized eggs extracted from female moths to high temperatures. This is achieved by the method. All individuals hatched from these unfertilized eggs have the same genetic makeup as their parents. It becomes a clone.

[0010] In general, it is difficult to freeze and preserve silkworm eggs using liquid nitrogen, but it is possible to do so using ovaries. Cryopreservation techniques have been established (Mochida Y., et al., 2007, J Insect Biotech Sericol 7 6, 97-100; Kusuda J., et al., 1985, J Insect Physiol, 31(12): 963-967). claw Since silkworms can develop into individuals from unfertilized eggs, the silkworms collected from the cloned silkworms By freezing the ovaries, the cloned strain can be preserved for a long period of time. After thawing the frozen ovaries, they are transplanted into silkworms, which are then raised to adulthood. The only way to induce embryonic development is to subject unfertilized eggs to high temperature treatment. All of the above problems can be solved by creating clonal lines.

[0011] However, this solution also has a major problem. First, the frequency of parthenogenesis is low in normal lines. In addition, existing established parthenogenetic silkworm strains enter diapause in the egg stage to overwinter. It is a univoltine strain and lays only diapause eggs. The eggs used in this process are non- Therefore, in order to obtain genetically modified cloned silkworms, it is necessary to use existing methods. Non-diapause eggs produced by ovarian transplantation of parthenogenetic strains are used, or parthenogenetic strains that lay non-diapause eggs are used. It was necessary to use the developmental lineage.

[0012] In order to solve the above problem, the present inventors transplanted the ovaries of parthenogenetic strains into male silkworms. We have developed a method to produce non-diapause eggs (Zabelina V. et al., 2015, J. Insect Physiol., 81 28-35). The diapause eggs of silkworms are formed when the diapause hormone synthesized in the female parent body acts on the developing ovaries. However, because males do not express diapause hormone, the eggs in the transplanted ovaries do not enter diapause. All eggs are non-diapause, without being affected by diapause hormones. However, with this method, The hatching rate of non-diapause eggs is very low, which makes it extremely difficult to produce transgenic cloned silkworms. The problem was that it was very low.

[0013] The present inventors continued their research and produced a parthenogenetic silkworm line that lays non-diapause eggs. Using this strain, they developed a new method for producing transgenic silkworms (Zabelina V. (E. et al., 2018, J. Biotechnol. Biomaterials, 8:24). In this method, as shown in Figure 1A, First, we investigated the dormant parthenogenetic silkworm strains that are capable of parthenogenesis and lay dormant eggs, and the dormant parthenogenetic strains that have a high parthenogenetic rate. The silkworms are then crossed with non-diapause strain silkworms that produce non-diapause eggs at low levels to produce F1 strains. and obtaining female F1 individuals, or F2 individuals by inbreeding the F1 individuals. In this F1 or F2 female population, a certain proportion of parthenogenetic lines (non-diapause) lay non-diapause eggs. Therefore, unfertilized eggs were collected from F1 or F2 female individuals, and parthenogenetic lines were By repeating the development process, non-dormant lines with high parthenogenetic potential are selected. The unfertilized eggs of the non-diapause parthenogenetic strain were subjected to parthenogenetic treatment, and the eggs that began to develop were Genetic recombination is then performed. After that, the target gene is selected. It is possible to produce recombinant cloned silkworms. However, this method requires crossbreeding and selective breeding. As mentioned above, there are many complicated and time-consuming steps involved, and it takes a lot of time and effort to make them. In addition, the hatching rate is reduced by introducing genes into unfertilized eggs. However, this method also entails the problem of lowering the efficiency of producing the desired genetically modified cloned silkworms. Therefore, it is difficult to say that this method is suitable for practical use. [Means for solving the problem]

[0014] Therefore, the present inventors have attempted to create a parthenogenetic silkworm strain that lays non-diapause eggs in a simpler manner. We also worked on developing technology to improve the hatching rate after gene transfer. As in 1B, genes are introduced into fertilized F1 eggs, and unfertilized eggs are collected from the F1 female individuals after hatching. The cells are then taken to induce parthenogenesis, and genetically modified individuals are selected from the resulting clones. We have succeeded in creating a new, unprecedented method for producing cloned transgenic silkworms. According to the study, the hatching rate of eggs after gene transfer is higher than that of the conventional method, and the number of steps is reduced to one. Because the generation time is shorter, it is possible to produce the desired genetically modified cloned silkworms in a short period of time. The present invention is based on the results of this development and provides the following:

[0015] (1) A method for producing genetically modified cloned silkworms, which involves crossbreeding two strains of silkworms. a mating step, an egg collection step in which female individuals are allowed to lay eggs after the mating step, and fertilized eggs obtained in the egg collection step. a nucleic acid introduction step for introducing a nucleic acid of interest into an egg; a female individual obtained from the fertilized egg after the nucleic acid introduction step; a parthenogenesis induction step of subjecting unfertilized eggs of the organism to parthenogenesis induction treatment, and A selection process in which genetically modified individuals are selected as genetically modified cloned silkworms from the individuals generated in the process. The method for producing said product comprises the steps of: (2) In the mating process, one or both of the two strains of silkworms lay non-diapause eggs. The method for producing silkworms according to (1), wherein the silkworms are silkworms that produce silkworms. (3) Silkworms that lay non-diapause eggs are those that have been developed from eggs that have been treated for laying non-diapause eggs. The method for producing the mouse according to (2), wherein the mouse is a female. (4) The method for producing the non-dormant oocytes described in (3), wherein the non-dormant oocyte pre-treatment is a low-temperature dark blueing treatment. (5) The parthenogenetic induction treatment is a high temperature treatment in which unfertilized eggs are exposed to 45°C to 50°C for 15 to 20 minutes. The method for producing a semiconductor device according to any one of (1) to (4), (6) A method for producing a recombinant cloned silkworm according to any one of (1) to (5) above. Genetically modified cloned silkworms. (7) A method for preserving a genetically modified cloned silkworm, comprising the steps of: an ovariectomy step of removing ovaries from female cloned silkworms; The preservation method further comprises a freezing step of freezing the isolated ovaries at -80°C or below. (8) The female individual of the genetically modified cloned silkworm is at least one of the 3rd to 5th instar larvae. The storage method according to (7), (9) The genetic material obtained by the method for preserving the genetically modified cloned silkworms described in (7) or (8) Frozen ovaries of cloned recombinant silkworms. [Effects of the Invention]

[0016] According to the method for producing genetically modified cloned silkworms of the present invention, the production can be carried out simply and efficiently with a small number of steps. This makes it possible to efficiently produce genetically modified cloned silkworms. According to the method for preserving genetically modified cloned silkworms of the present invention, This will enable stable maintenance and propagation of the strain, and also make its management easier. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a conceptual diagram of a method for producing genetically modified cloned silkworms when diapause-producing parthenogenetic silkworms and non-diapause-producing silkworms are used as parent silkworms. A shows the conventional method, and B shows the production method of the present invention. [Figure 2]In this figure, the mating step (S201) to the selection step (S206) are a flow diagram of the method for producing genetically modified cloned silkworms of the present invention, and the mating step (S201) to the freezing step (S208) are a flow diagram of the method for preserving genetically modified cloned silkworms of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] 1. Method for producing genetically modified cloned silkworms Overview The first aspect of the present invention is a method for producing a transgenic cloned silkworm. The target nucleic acid is introduced into the F1 fertilized eggs obtained by crossbreeding the silkworm strains, and the grown F1 female adults are Unfertilized eggs collected from the body are subjected to parthenogenesis induction treatment, and the resulting cloned silkworms are transformed. This method requires less labor and does not require ovarian transplantation or F2 as in the conventional method. This method makes it possible to efficiently produce genetically modified cloned silkworms in a short period of time. The use of fertilized eggs facilitates gene transfer procedures and improves hatching rates.

[0019] 1-2.Definition of Terms The following terms frequently used in this specification are defined below. "Genetic modification" refers to the artificial modification of the natural genetic information of a host organism. The modification of genetic information referred to here includes addition, deletion, substitution, etc. of genetic information. Artificial modification of information can be achieved by existing genetic engineering techniques, such as plasmids. A method of adding genetic information that is not possessed by the host organism using vectors or transposons such as or a method for destroying the genetic information possessed by the host organism, or a method for modifying the host organism by genome editing Examples of methods include modifying the genome information of a gene.

[0020] "Genetically modified silkworms" refers to silkworm genes produced using the above-mentioned genetic engineering technology. The term "genetically modified silkworms" as used herein refers to recombinant silkworms, or their progeny. A genetically modified organism obtained by introducing foreign DNA into silkworm eggs using the clonal injection method. say.

[0021] "Clone silkworms" are individuals with the same genome composition and genetic makeup. Generally, unfertilized eggs obtained from parthenogenetic silkworms, which will be described later, are used. The silkworm population obtained by inducing parthenogenesis on silkworms is called a clonal silkworm.

[0022] As used herein, the term "genetically modified cloned silkworms" refers to genetically modified cloned silkworms. In the case of genetically modified cloned silkworms, the foreign DNA introduced is also cloned. It is identical among individuals in the population. Therefore, each individual of the transgenic cloned silkworm In principle, the quality and expression level of the protein encoded by the foreign DNA produced in the

[0023] As used herein, a "lineage" refers to individuals that share specific genetic traits within the same species. A population of individuals, roughly the same concept as a "strain." A mutant with a specific gene mutation In this specification, the term "line" also includes groups and varieties that share common morphology or characteristics. Therefore, unless otherwise specified, the biological species in this specification The lineage in the silkworm breeding program is called a "silkworm lineage," and silkworm individuals belonging to such a lineage are called "lineage lineages." For example, the diapause strain described below is a diapause strain of silkworms, i.e., a diapause strain of silkworms. The silkworms belonging to this dormant silkworm lineage are called "dormant silkworms."

[0024] When focusing on different genetic traits, an individual can belong to multiple lineages. For example, diapause parthenogenesis The developmental lineage has two genetic traits, dormancy and parthenogenesis, so if we focus on dormancy, It belongs to a dormant lineage, and if we focus on parthenogenesis, it belongs to a parthenogenetic lineage.

[0025] In this specification, "dormancy" refers to a state in which an organism ceases to develop or grow at a specific time in its life cycle. or to temporarily stop activities and enter a dormant state.

[0026] As used herein, the term "diapause (silkworm) strain" refers to a strain that is related to a specific genetic trait called egg diapause. In this specification, "spawning" is often referred to as "laying under" ("spawning under"). Silkworms are divided into univoltine, bivoltine and multivoltine strains. Many of the voltinite strains are dormant silkworm strains that lay dormant eggs. When reared under the specified conditions, the strain produces adults once a year. A "multivoltine lineage" is a lineage in which adults emerge multiple times a year. Generally, many of the derived lineages that have an ancestral lineage in temperate regions where winter occurs are univoltine lineages. However, even in the case of diapause silkworm strains, non-diapause eggs can be produced by the non-diapause production pretreatment described below. Specific examples of diapause silkworm strains include, but are not limited to: ,For example, Daizo, No. 137, No. 146, No. 603, No. 604, No. 604, No. 605, No. 514, Naka No. 515, Naka No. 9.0, Nihon No. 9.0, Shunrei, Zhongyue, Hitachi, Nishiki, Nihon No. 502, Branch No. 146, Branch No. 122 , Tokushi No. 2, Ou No. 7, Tokushi No. 4, and Kakushina, etc.

[0027] "Diapause eggs" are eggs obtained by laying diapause silkworm strains under normal breeding conditions. In principle, they enter a state of diapause during the egg stage. In diapause eggs, embryonic development usually stops at the embryonic stage, and they are tolerant to low temperatures. This is a life cycle control phenomenon based on the environmental response that silkworms acquire in order to survive the year. In nature, diapause of diapause eggs occurs when the accumulated temperature at 5°C reaches a certain value. This will release the setting.

[0028] As used herein, the term "non-diapause (silkworm) strain" refers to a non-diapause silkworm strain that does not enter a diapause state during the egg stage. It refers to a population of individuals that lay eggs. Generally, they have ancestral lineages in subtropical or tropical regions where there is no winter. Many of the derived strains are polyvoltine silkworm strains that repeat multiple generations per year, and these are non-diapause strains. It is a non-diapause silkworm strain that lays eggs. Specific examples of non-diapause silkworm strains include, but are not limited to: However, some examples include Mysore, Nistari, Pure Mysore, Annan, and Ringetsu.

[0029] As used herein, the term "non-diapause eggs" refers to silkworm eggs that do not enter a diapause state. Specific examples include non-diapause eggs and non-diapause eggs obtained from bivoltine or multivoltine silkworm strains. Examples include non-diapausing eggs obtained from female individuals of treated diapausing silkworm strains.

[0030] As used herein, "non-diapause egg production treatment" refers to the treatment of diapause silkworm strains that normally produce diapause eggs. It refers to the process of applying a specific treatment to female individuals to induce them to lay non-diapause eggs. A specific example of the specific treatment is the low-temperature dark blueing treatment described in JP 2017-085958 A.

[0031] In this specification, "incubation" refers to the process of incubating silkworm eggs at an appropriate temperature to ensure uniform development of the silkworms. By controlling the humidity and light conditions, the hatching of multiple silkworm eggs can be made uniform. cormorant.

[0032] As used herein, the term "low-temperature dark germination treatment" refers to germination treatment carried out under low-temperature dark conditions. By subjecting eggs of the silkworm strain to a low-temperature dark incubation treatment, the number of female individuals hatched from the eggs is increased. Some of the eggs that are released will be non-diapause eggs.

[0033] As used herein, the term "parthenogenetic (silkworm) line" refers to a line in which parthenogenesis is induced with high efficiency. Generally, most of the silkworm strains that are capable of parthenogenesis are diapause silkworm strains. Therefore, in this specification, the parthenogenetic silkworm strain refers to a parthenogenetic silkworm strain unless otherwise specified. "Diapause parthenogenetic (silkworm) strain" is both a developmental and a diapause silkworm strain. The parthenogenetic silkworm strains are all produced by applying physical or chemical stimuli to unfertilized eggs. Although the parthenogenetic silkworm strain is not limited to this, for example, PK1 Examples include the P14 line and the Camboge x Hi105 hybrid.

[0034] 1-3.Production method The flow of the method for producing the genetically modified cloned silkworm of the present invention is shown in Figure 2. As described above, the production method of the present invention includes a mating step (S201), an egg collection step (S202), and a nucleic acid introduction step (S203). The unfertilized egg collection process (S204), the parthenogenesis induction process (S205), and the selection process (S206) are essential steps. Each step will be explained below.

[0035] (1)Mating process The "mating process" (S201) is a process in which two strains of silkworms are mated. It is sufficient if the female lays non-diapause eggs and the next generation of F1 females can produce parthenogenetic unfertilized eggs. A preferred combination is one in which the silkworm is of the Japanese or European species and the other in which the silkworm is of the bivoltine species. It is a Chinese strain of silkworm. Specifically, it is a combination of Japanese 9.0 strain silkworm and Chinese 9.0 strain silkworm. Examples of lines in which F1 females lay non-diapause eggs include, but are not limited to, In addition to the naturally non-diapause silkworm strains such as the voltinism strains, Examples include diapause silkworm strains that have undergone non-diapause birth pretreatment, such as injection of Female individuals that develop from eggs that have undergone the diapause procedure can also lay non-diapause eggs. However, female parents developed from the treated eggs can be used as non-diapause silkworms. If the female individual is a non-diapause strain silkworm, the other male individual may be a diapause strain or a non-diapause strain. Any system can be used regardless of the type.

[0036] The non-dormant germination treatment may be, for example, a low-temperature dark germination treatment. For example, eggs can be kept in the dark at a low temperature of 15°C for about a month, and then allowed to develop slowly. By carrying out this treatment on fertilized eggs of diapause silkworm strains, Some of the eggs laid by the female silkworms derived from the fertilized eggs will be non-diapause eggs. Regarding the blue method, see Eiichi Ozegawa et al. (2000, Nihon Sanzatsu, 69(6): 369-375) or Isamu Shimizu (1991, The low-temperature dark incubation method is a method that physically damages the eggs. The method is advantageous in that it allows the production of non-diapause eggs without the need for a low-temperature dark incubation treatment. Examples of silkworm strains include Shi146 and Zhong510. Silkworm mating can be carried out according to the usual method. Generally, females are able to mate immediately after hatching. Mating lasts for 2 to 3 hours, and mating once is usually sufficient.

[0037] (2) Egg collection process The "egg collection step" (S202) is a step in which the female individuals after the mating step are allowed to lay eggs. In this step, female silkworms are used after mating, so the eggs collected in this step (S202) are, in principle, The egg collection method is not limited. In the art, the egg is usually collected from female silkworms after mating. The method used is to provide a spawning mat and allow the fish to lay eggs on the mat. When eggs are given a spawning mat, they often start laying eggs within a few hours. In order to efficiently incorporate the target nucleic acid introduced into the fertilized egg in the introduction step (S203) into the nucleus, It is desirable to start nucleic acid transfer within 2 to 8 hours, preferably 3 to 6 hours, after In this step, it is preferable to allow the female individual to give birth in accordance with the timing of the nucleic acid introduction. The mating male and female silkworms or the mating female silkworms are placed under a low temperature of 0 to 10°C, preferably 5°C, After storing at that temperature for 1-2 days, or at most 1-7 days, the female silkworms are transferred from the low temperature to room temperature (23-28 This can be achieved by transferring the eggs to a dark room (20°C), providing them with egg-laying mats, and allowing them to start laying eggs under dark conditions. Silkworm embryonic development begins immediately after birth, so this method allows for the embryonic development stage to be completed in this process. However, it is possible to stably obtain fertilized eggs at a stage suitable for nucleic acid transfer.

[0038] (3) Nucleic acid introduction step The "nucleic acid introduction step" (S203) is a step of introducing a nucleic acid of interest into the fertilized eggs obtained in the egg collection step (S202). Some of the fertilized eggs to be introduced with nucleic acids are non-diapause eggs. It is desirable to introduce the nucleic acid into multiple fertilized eggs obtained in (S202).

[0039] In this process, the "nucleic acid of interest" refers to the nucleic acid that is used to produce the desired genetically modified silkworms. The nucleic acid to be introduced into the silkworm is usually DNA, and preferably the desired nucleic acid. Incorporation into the genome of genes, expression vectors containing the genes, and helper plasmids When introducing a gene, the type of gene does not matter. For example, a protein It may be a gene encoding a desired gene that can confer a desired trait, or a gene encoding a functional nucleic acid (such as an RNAi molecule). Genes can be introduced. The origin of the gene does not matter. It is derived from the host silkworm. It may be a gene derived from a living organism of another species, for example, a human. When producing antibodies using genetically modified silkworms, the foreign DNA is It can be an expression unit containing gene expression regulatory regions such as promoters and terminators. do.

[0040] This step can be carried out by a method known in the art for introducing a foreign gene into silkworms. For example, if the expression vector contains the inverted terminal repeats (HTAs) of a transposon at both ends of the target DNA, ndler AM. et al., 1998, Proc. Natl. Acad. Sci. USA 95:7520-5) In the case of mido, the method of Tamura et al. (Tamura T. et al., 2000, Nature Biotechnology , 18, 81-84), and the method of Zhou et al. (Zhou W. et al., 2012, Insect Science, 19: 172-182) Specifically, water or a buffer solution can be used to adjust the concentration of the expression vector to an appropriate level. The injection is prepared by dissolving or diluting the drug in a solvent such as a fluoride. A helper plasmid containing DNA encoding a sposon transferase is added. An example of a per-plasmid is pHA3PIG.

[0041] As described above, the nucleic acid is introduced into the fertilized eggs obtained in the egg collection step (S202) 3 to 6 hours after laying. The injection is carried out by microinjection into the cells. Generally, a special injection device that uses air pressure is used. For example, Patent No. 1654050 or T The method of Tamura et al. (Tamura T, et al., 2007, J Insect Biotechnol Sericol, 76: 155-159 The amount of nucleic acid to be introduced is not particularly limited. The amount can be determined appropriately depending on the situation. It is usually 1 nL to 5 nL. After nucleic acid introduction, silkworm eggs are incubated for a period of The mixture may be incubated at 100°C under suitable conditions, for example, at 25°C.

[0042] (4) Unfertilized egg collection process The "unfertilized egg collection step" (S204) is a step of collecting unfertilized eggs from female individuals. In this way, the female individual is an individual in which an unfertilized egg has developed to a state where parthenogenesis is possible within the body. Preferably, they are adults, more preferably, they are kept at 0 to 10°C, preferably immediately after emergence or within 2 days after emergence. The adult insects should be refrigerated at a low temperature of 5°C for no more than 10 days.

[0043] There are no limitations on the method of collecting unfertilized eggs. For example, they can be collected by dissection or by natural spawning. The method of collection includes:

[0044] Specifically, the method of collecting by dissection involves, for example, cutting the abdomen or tail of a female adult with a scalpel or dissection knife. After making an incision with dissection scissors, pressure is applied to the abdomen with the fingers to push the ovaries out through the incision. The clumped fallopian tubes can be loosened by immersing the removed ovaries in water. Next, place the ovaries on a fine stainless steel mesh or gauze and rub them with your fingers. This separates the oviduct tissue from the unfertilized eggs. Finally, the water is poured through a mesh to separate the oviduct tissue from the unfertilized eggs. The oviduct tissue floating on the surface is removed, and the unfertilized eggs remaining at the bottom are collected. This procedure can be repeated several times. This allows only unfertilized eggs to be collected.

[0045] The natural egg collection method involves having unmated female silkworms lay unfertilized eggs after hatching. A specific method for causing virgin female silkworms to lay unfertilized eggs is to use the egg collection method described above. The method described above may be followed.

[0046] (5) Parthenogenesis induction process The "parthenogenesis induction step" (S205) is a step of inducing parthenogenesis in the unfertilized eggs obtained in the unfertilized egg collection step (S204). This is a step of carrying out a parthenogenetic induction treatment. The parthenogenetic induction treatment can be carried out according to a method known in the art. For example, the method described by Sugai et al. (1983, Journal of Japanese Sericultural Science, Vol. 5) 2, No. 1: 51-56), Hirokawa (Hirokawa Masahiko, 1990, Fukushima Silkworm Experimental Research Institute, 24: 1-6), and Ozegawa (Ko Segawa E., et al. 2012, J Insect Biotechnol Sericology, 81: 37-44) Generally, unfertilized eggs are subjected to physical or chemical stimulation. Parthenogenesis is induced by applying high temperature treatment. As a specific example of heat treatment, the unfertilized eggs collected in the above process are treated with hot water at 45°C to 50°C for 15 minutes. After that, it is preferable to keep it at 12 to 18°C ​​for 2 to 4 days. .

[0047] (6) Selection process The "selection process" (S206) is a process of selecting genetically modified silkworms from silkworms hatched after the parthenogenesis induction process. This is the process of selecting the genetically modified cloned silkworms. This step may also be carried out by a method known in the art. If the vector contains a marker gene, the desired genetic modification can be identified based on the expression of the marker gene. In this case, selection is based on the expression of marker genes, etc. The resulting silkworms become genetically modified cloned silkworms.

[0048] A "marker gene" is a base sequence that encodes a marker protein, also known as a selection marker. It is a polynucleotide consisting of:

[0049] "Tagged protein" refers to a protein that confers new traits not present in the host silkworm by expressing a tagged gene. Proteins that can be synthesized, such as enzymes, fluorescent proteins, pigment synthesis proteins, or luminescent proteins Based on the activity of the labeled protein, a transformant carrying the introduced nucleic acid can be identified. Here, "based on activity" means that the activity can be easily determined based on the detection result of the activity. The activity detection is based on the activity of the labeled protein itself. The detection may be by detection of the protein itself or indirectly via metabolites generated by the protein's activity. The detection may be chemical detection (including enzyme reaction detection), physical detection, or the like. detection (including behavioral detection), or sensory detection by the detector (including vision, touch, smell, hearing, detection by taste).

[0050] The type of labeled protein may be any of the following, as long as its activity can be detected by a method known in the art: There are no particular limitations. Preferably, a host carrying a marker for distinguishing a transformant is used for detection. That is, it is a labeling protein that is less invasive to the transformant. For example, fluorescent proteins , pigment synthesis proteins, photoproteins, exocrine proteins, proteins that control external morphology Fluorescent proteins and pigment-synthesizing proteins are used to detect the external appearance of transformants. It can be visually detected under certain conditions without changing the It is particularly suitable because it is very low invasive and the identification and selection of transformants is easy.

[0051] As used herein, a "fluorescent protein" refers to a protein that emits light of a specific wavelength when irradiated with excitation light of a specific wavelength. It may be either a natural or non-natural type. The excitation wavelength and the fluorescence wavelength are not particularly limited. Specifically, for example, CFP, RFP, DsRed (DsRed -monomer), YFP, PE, PerCP, APC, GFP (including derivatives such as EGF) etc.

[0052] As used herein, the term "pigment synthesis protein" refers to a protein involved in the biosynthesis of a pigment. The term "pigment" used here refers to a substance that can give a pigment to a transformant. It is a low molecular weight compound or peptide that can be used for the external color of the individual, regardless of its type. For example, melanin pigments (including dopamine melanin), omokro Examples of suitable dyes include rhodium-based dyes and pteridine-based dyes.

[0053] 1-4. Genetically modified cloned silkworms By the production method of the present invention, genetically homogeneous mutants having the same genome composition containing the gene of interest can be produced. High-quality transgenic cloned silkworms can be obtained.

[0054] The transgenic cloned silkworm of the present invention is a parthenogenetic silkworm that has an exogenous gene in its genome. This genetically modified cloned silkworm is produced by the production method of the present invention. It is possible to clone the unfertilized eggs and then pass them on as clones. The genetic factors that cause parthenogenesis are unclear.

[0055] As mentioned above, the genetically modified cloned silkworm of the present invention is a parthenogenetic silkworm strain. Therefore, unfertilized eggs were collected from the female individuals of the genetically modified clones obtained, and parthenogenetic development was carried out. By carrying out the induction process, it is possible to obtain multiple clones of genetically homogeneous transgenic silkworms. Furthermore, the newly obtained transgenic clones were obtained by parthenogenesis induction treatment. Unfertilized eggs were collected from female Equine fish and subjected to the same parthenogenesis induction treatment to induce the genes. It will also be possible to maintain the lineage of recombinant silkworms and mass-produce cloned silkworms.

[0056] 1-5.Effects According to the method for producing genetically modified cloned silkworms of the present invention, the production process requires fewer steps than conventional methods. To efficiently produce clones of transgenic silkworm strains in a short period of time with a high hatching rate. This will reduce the cost of producing genetically modified silkworm clones and By mass-producing the clone, it is possible to mass-produce proteins of stable quality. This makes it possible to produce the product quickly and cheaply.

[0057] 2. Preservation method of genetically modified cloned silkworms 2-1. Overview A second aspect of the present invention is a method for preserving genetically modified cloned silkworms. According to the method, the ovaries extracted from the female individuals of the genetically modified cloned silkworms obtained in the first embodiment By storing them at ultra-low temperatures, it is possible to maintain genetically modified cloned silkworm strains through crossbreeding. This makes it possible to preserve the clones for long periods without the need for further maintenance.

[0058] 2-2. Method The flow of the preservation method of the present invention is shown in Figure 2. In Figure 2, the genetically modified clone of the first embodiment is In the silkworm production method flow, the genetically modified silkworm obtained after the selection process as a derivative flow In order to maintain the cloned silkworm lineage, the method for preserving the cloned silkworm is applied. Therefore, the preservation method of this embodiment includes the previous steps in the flow of FIG. 2, but does not include the mating step (S2 The steps 01) to 206) of the selection process are the same as those of the first embodiment of the method for producing a recombinant cloned silkworm. Therefore, the ovary extraction process (S207) and the freezing process (S208) of the cloned silkworms are characteristic processes of this embodiment. Therefore, these two steps will be explained below.

[0059] (1) Clonal ovary removal process The "clone ovary removal step" (S207) is a step for producing the first embodiment of the genetically modified cloned silkworm. The method comprises the step of extracting ovaries from female individuals of genetically modified cloned silkworms obtained by the method. The female individuals that are the targets of ovariectomy in this process are the first genetically modified cloned silkworms. The cloned silkworms may be the first generation (G1: Generation 1) produced or the untransfected silkworms of the cloned silkworms. The second generation (G2) or later developed from the fertilized eggs may be used. The stage may be a larval stage, a pupal stage, or an adult stage. Preferably, the larval stage, more preferably the third instar or is the fourth instar larva.

[0060] The basic operation in this step may be in accordance with a method known in the art and is not particularly limited. For example, Zabelina et al., 2015 (mentioned above) or Yoko Takemura and Yuji Mochida (Silkworm and Insect Biotechnology) 2008, 77(1):9-16). The ovaries collected in this study are all derived from genetically modified organisms containing foreign nucleic acids of interest. be.

[0061] (2) Freezing process The "freezing step" (S208) involves freezing the ovaries obtained in the cloned ovary extraction step (S207) at an ultra-low temperature. The ovaries may be frozen according to methods known in the art. For example, the ovaries extracted from the female larvae of the genetically modified cloned silkworm were cultured in an insect cell culture medium containing 1.5 M DMSO. A plastic tube containing medium for cell culture (e.g., Grace's Insect Medium: Lonza) After freezing in a refrigerator, the ovaries were stored in liquid nitrogen as ovaries for genetically modified cloned silkworm strains. The freezing method and freezing temperature are ultra-low temperatures, for example, -80°C or lower, preferably -89°C or lower. The freezing method is not particularly limited as long as it can be frozen under the conditions below. One method is to freeze the ovaries of the parrotfish using liquid nitrogen vapor.

[0062] 2-3.Frozen ovaries The frozen ovaries obtained by the above-mentioned method for preserving genetically modified cloned silkworms can be used for a long period of time. It is possible to store it at extremely low temperatures.

[0063] The method for recovering cloned silkworms from frozen ovaries may be in accordance with methods known in the art. For example, frozen ovaries, preferably the entire freezing container, are thawed by immersion in warm water, and then the ovaries are transferred to a host. The transplanted ovaries are then transplanted into other silkworm larvae that will become the host. After raising the transplanted silkworms to adulthood, The extracted unfertilized eggs are then subjected to parthenogenesis induction treatment such as high temperature treatment. By carrying out this process, the frozen cloned silkworms were awakened and re-emerged. The parthenogenesis induction treatment can be carried out by the parthenogenesis induction step (S205) described in the first embodiment. ) method. By raising the individuals that hatch from the eggs, the desired genetic modification can be Cloned silkworms can be recovered.

[0064] 2-4.Effects The method for preserving genetically modified cloned silkworms of the present invention and frozen ovaries obtained thereby According to the study, the genetically modified silkworm strains produced can be maintained as clones for a long period of time. This makes it possible to avoid the need for crossbreeding and facilitates maintenance and genetic variation. There is no difference. [Example]

[0065] In order to clarify the present invention, the following examples will be given to explain the present invention in more detail. The examples are intended to illustrate one embodiment of the present invention and are not intended to limit the scope of the present invention. isn't it.

[0066] <Example 1: Production of genetically modified cloned silkworms> (the purpose) The method for producing genetically modified cloned silkworms of the present invention is The occurrence rate of non-diapause eggs in F1, the hatching rate of eggs after gene introduction, and the number of transformants were measured. The yield was verified.

[0067] (method) (1) Gene transfer into F1 fertilized eggs Two strains of silkworms were crossed to obtain eggs of non-diapause parthenogenetic strains. As a lineage, male silkworms were used as diapause parthenogenetic silkworm lineage (day 9.0), and female silkworms were used as diapause parthenogenetic silkworm lineage (day 9.0). The female silkworms used here were kept in a low-temperature, dark environment during their egg stage. They have been treated with the hyphal-inducing method, and although they are a diapause silkworm strain, they lay non-diapause eggs. The blue treatment was carried out by incubating the fertilized eggs at a low temperature of 15°C. Some eggs obtained by the conventional low-temperature dark incubation method become non-diapause. This has been demonstrated in Japanese Patent Application Publication No. 2017-085958 by the present inventors.

[0068] Next, the female silkworms that emerged were mated with the male silkworms for mating for 3 hours, and then the male and female parents were mated. Immediately store the tube in a refrigerator (4°C) overnight while the target nucleic acid is being introduced. Preparations were made for the event.

[0069] The recombinant DNA solution for microinjection was prepared using the HiSpeed ​​Plasmid Midi Kit (Qiagen). When ready for microinjection, mated females were The parent silkworms were taken out of the refrigerator and allowed to lay eggs on glue-coated egg-laying mats, and F1 fertilized eggs were collected.

[0070] F1 fertilized eggs 0-2 hours after spawning were fixed on a slide glass with adhesive, and then the target gene was transfected. pBac3xP3DsRed (160 ng / μl) was mixed with transposon transferase mRNA (80 ng / μl) and the enzyme The DNA was mixed with helper DNA (160 ng / μl) and introduced by microinjection. After microinjection, the eggs were incubated on the slide glass in a humidified environment at 25°C until hatching. After that, the number of hatched F1 larvae was counted and the number of microinjected F1 larvae was The hatchability was calculated from the number of eggs that hatched relative to the number of eggs introduced (number of eggs introduced). The results are shown in Table 1.

[0071] [Table 1]

[0072] In the table, experimental plots Y179 and Y180 are plots where the same experiment was conducted independently. The percentage in parentheses for the number of non-diapausing eggs indicates the ratio to the number of eggs introduced. The percentage indicates the hatching rate. The number of moths from which eggs were collected was determined by rearing the hatched individuals and then collecting the eggs in (3) described below. The number of moths containing TG eggs is the number of moths that were born as a result of gene transfer among the female adults from which eggs were collected. The number of moths containing transgenic eggs (TG eggs) is shown.

[0073] Obtained by mating male individuals of a diapausing silkworm strain with diapausing female individuals that lay non-diapausing eggs. It was shown that about 50% of F1 eggs become non-diapause eggs. A high hatching rate of approximately 40% was also confirmed for the F1 eggs. This hatching rate was higher than that of the conventional method shown in Figure 1A. hatching rate (14-18%) (Zabelina V. et al., 2018, J. Biotechnol. Biomaterial s, 8:24;Zabelina V. et al., 2019, The 25th International Congress on Sericultur e and Silk Industry (Proceedings), 2019, p93) This is what happened.

[0074] (2) Growth of F1 individuals The F1 larvae hatched in each experimental group were transferred to a container serving as a silkworm bed using a feather broom. For rearing larvae, plastic lunch boxes (DX-HS8: 222 × 143 × 35 mm; or DX-HS20: 25 The feed was Silkmate original species 1-3 S (Nippon Agricultural Co., Ltd.). The food was generally changed once for the first and second instars, once for the third instar, and once for the third instar. This was done 1 to 3 times, and when the food was insufficient or dried out, it was replaced as needed. If there was a lot of leftover food, it was removed, but if there was only a little, it was left as it was and new artificial feed was introduced. Added.

[0075] For rearing 4th to 5th instar silkworm larvae, use seedling boxes for horticulture, type 2 (product number 201206; Sanko Co., Ltd.). Use a container lined with silkworm paper (craft paper), dry-proof paper (paraffin paper), or both. Depending on the humidity and conditions inside the container, the container may be covered with a dry-proof paper, acrylic, or mesh lid. After the third day, the number of animals in each container is counted, and the number is set at approximately 80 animals or less per container, depending on the size of the container. The food was the same as for the larval stage of young silkworms, with Silkmate original species 1-3 years old M (Nihon Nosan Kogyo) in strips. The food was cut into strips or cut into pieces and given to the animals. The timing of food replacement varies depending on the amount of leftover food and the dryness of the food. In principle, this was done once a day. If there was only a small amount of food left, the old food was not removed and the remaining food was left. Fresh artificial feed was added by cutting. The rearing temperature was 28°C for 1st to 4th instars and 25°C for 5th instars. The mature silkworms are transferred to the husks (jozok: transferring the larvae to the husks) and then left for 25 days. After the cocoons are made at 300°C, the cocoons are cut open to remove the pupae and placed in moth boxes (cardboard boxes used to hold pupae and moths). The eggs were placed in a protective box (like a box) and protected until they emerged.

[0076] (3) Collection of unfertilized eggs from F1 female individuals and induction of parthenogenesis Unfertilized eggs were collected from emerged F1 females by natural spawning and dissection. The female adults containing transformed eggs obtained from the experimental plot Y179 in Table 1 were refrigerated without mating. After storing in a refrigerator (4°C) for 1-7 days, transfer to room temperature (25°C), attach a spawning mat, and allow unfertilized eggs to be laid. I did.

[0077] On the other hand, the female adults containing transformed eggs obtained from the experimental plot Y180 in Table 1 had tails after emergence. The ovaries were then removed and the unfertilized eggs were extracted. The oviducts were then unwound and the ovaries were placed in a mesh with a mesh size of 0.5 mm or less. The ovaries were placed on a stainless steel mesh and drained. The oviduct tissue and unfertilized eggs were separated by pouring water over the stainless steel mesh. The floating oviduct tissue was removed, and the unfertilized eggs that had sunk to the bottom of the container were collected. This procedure was repeated several times. By doing this, only unfertilized eggs were prepared.

[0078] Next, unfertilized eggs obtained from each experimental group were immersed in hot water at 46°C for 18 minutes. The mixture was then cooled in 15°C cold water for 30 minutes, air-dried, and stored at 15°C for 3 days. Unfertilized eggs are immersed in hydrochloric acid with a specific gravity of 1.11 for 1 hour, and then artificial hatching is performed by immediate acid immersion. It turned blue.

[0079] (4) Selection of transformants (genetically modified organisms) Transformation by gene transfer from the first generation clone (G1) developed parthenogenetically from F1 unfertilized eggs The clones that hatched were selected (TG clones: transgenic clones). The genetically modified plants were grown in accordance with the procedure described in "(2) Growth of F1 individuals." The eggs and larvae exhibit a phenotype in which the ocelli of the eggs and larvae exhibit red fluorescence. The results are shown in Table 2.

[0080] [Table 2]

[0081] The experimental plots Y179-1 and Y180-2 in the table were obtained from the experimental plots Y179 and Y180 listed in Table 1, respectively. The percentage in parentheses for the number of TG clone moths is the number of moths surveyed. The figure shows the appearance ratio of TG clone moths in the oviposition. Regardless of the isolation method, the occurrence rate of TG clones was approximately 3%. Through the above procedures, the desired genetically modified cloned silkworms were obtained.

[0082] (5) Lineage development of transgenic cloned silkworms (4) The first generation (G1) of transgenic cloned silkworms obtained from each experimental group The transgenic plants (TG larvae) were screened at the larval stage. The results are shown in Table 3.

[0083] [Table 3]

[0084] The number of TG-collected moths was determined by raising TG clone larvae to adulthood and then collecting unfertilized eggs for G2 production. The number of female moths is shown.

[0085] The TG clone larvae of G1 collected from each moth area were raised to adulthood, and the female individuals were The second generation (G2) was obtained by collecting unfertilized eggs in the same way and allowing them to develop parthenogenetically. The parthenogenesis rate and hatching rate of the G2 eggs obtained are shown in Table 4.

[0086] [Table 4]

[0087] Some of the hatched G2 larvae were reared and the expression of the introduced gene was examined. Gene expression (red fluorescence expression) was confirmed. The results are shown in Table 5.

[0088] [Table 5]

[0089] As shown in Table 5, the transgene was stably maintained in parthenogenetic individuals. . Next, unfertilized eggs were collected from G2 females in the same manner as above, and the next generation (G3) was developed. The parthenogenesis rate and hatching rate of the silkworms were investigated. The results are shown in Table 6.

[0090] [Table 6]

[0091] It was shown that G3 also showed almost the same parthenogenesis rate and hatching rate as G2.

Claims

1. A method for producing a genetically modified non-diapause cloned silkworm, comprising: a mating step in which female silkworms developed from the non-diapause-treated eggs are mated with male silkworms of any strain; an egg collection step in which female individuals after the mating step are allowed to lay eggs; a nucleic acid introduction step of introducing a nucleic acid of interest into the fertilized eggs obtained in the egg collection step; an unfertilized egg collection step of collecting unfertilized eggs from female individuals obtained from the fertilized eggs after the nucleic acid introduction step; a parthenogenesis induction step in which the unfertilized eggs obtained in the unfertilized egg collection step are subjected to parthenogenesis induction treatment; and a selection step of selecting genetically modified individuals as genetically modified cloned silkworms from individuals generated after the parthenogenesis induction step. The method for producing the same.

2. The method of claim 1, wherein the non-dormant birth pre-treatment is a low-temperature dark blueing treatment.

3. A method for producing a plant according to claim 1 or 2, wherein the parthenogenesis induction treatment is a high-temperature treatment in which unfertilized eggs are exposed to 45°C to 50°C for 15 to 20 minutes.

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