Method for producing transgenic silkworms using diapause eggs

Abstract

To develop and provide a technique for making transgenic silkworms of commercial lines in a short time and with little effort.SOLUTION: A method for making transgenic silkworms includes a step for introducing an intended nucleic acid into an egg, followed by a step for breaking the dormancy of the dormant egg by non-thermal equilibrium plasma treatment.

Description

[Technical Field]

[0001] The present invention relates to a method for producing transgenic silkworms using diapause eggs. [Background technology]

[0002] In recent years, silkworms (Bombyx mori) have been attracting attention as a suitable host organism for mass production of proteins. Silkworms are insects that have long been used industrially to produce silk, and are capable of producing large amounts of silk thread in a short period of time from the cocoons produced by their larvae. Utilizing this characteristic, it has become possible to mass-produce useful proteins other than silk thread using genetic engineering technology. Some of these proteins are on the market as animal testing agents, and development of production technologies for active pharmaceutical ingredients for human pharmaceuticals is also underway.

[0003] To produce transgenic silkworms, the microinjection method is used, in which a transposon is used to inject a desired gene into eggs (Non-Patent Document 1). Silkworm eggs are fertilized within two hours after oviposition, after which a bare nucleus without a cell membrane, called a syncytium, repeatedly divides and migrates to the surface of the egg. The desired gene must be introduced into the eggs within this period, specifically, two to eight hours after collection of the fertilized eggs. This is because the efficiency of emergence of transformants drops significantly after this period, during which embryonic development progresses (Non-Patent Document 2). In other words, to produce transgenic silkworms using existing methods, it is important to introduce the desired gene into eggs within a specific time period after oviposition, while embryonic development is not yet complete.

[0004] Most silkworms are univoltine, meaning that adults typically emerge once a year. Univoltine silkworms lay diapause eggs, which then enter a state of diapause. This state of diapause persists even after microinjection, so even if the gene is introduced into diapause eggs, transformants cannot be obtained. Therefore, non-diapause eggs, which do not enter a diapause state, are used in the microinjection method. However, many non-diapause strains that lay non-diapause eggs are experimental strains with poor silk productivity and are unsuitable for industrial use. Therefore, in the past, it was necessary to perform genetic modification using microinjection with non-diapause eggs from a non-diapause strain, and then crossbreed the resulting genetically modified silkworms with a diapause-producing strain to develop a practical strain suitable for industrial use.

[0005] In order to solve the above problems, methods have been developed for obtaining diapause eggs or non-diapause eggs that do not enter a diapause state (often collectively referred to as "non-diapause eggs, etc." in this specification) from a practical diapause strain. For example, there is a diapause-breaking method in which external physical or chemical stimuli are used to prevent diapause eggs from entering a diapause state and thereby maintain embryonic development, or to awaken diapause eggs from a diapause state and induce embryonic development, and a non-diapause egg-laying method in which parent silkworms of a diapause strain are treated to cause non-diapause eggs to be laid.

[0006] Non-Patent Document 3 discloses a method of microinjecting dormant eggs that have not entered a diapause state, which are obtained by acid immersion, a method of breaking diapause. However, this method has the problem that the subsequent hatching rate drops significantly, to only 3.4-4.6%, because the eggs are subjected to a physical stimulus, microinjection, in addition to the chemical stimulus of acid.

[0007] The low-temperature dark incubation method involves incubating parent eggs under low-temperature, dark conditions, and then allowing the hatched parent silkworms to lay non-diapause eggs (Non-Patent Documents 4 and 5). However, the low-temperature dark incubation method has the problem that the number of non-diapause eggs laid varies significantly depending on the silkworm strain, and sometimes no non-diapause eggs are laid at all (Patent Document 1). Furthermore, when the non-diapause eggs obtained by this method are microinjected, there is also the problem that the hatching rate drops drastically, as with the acid immersion method.

[0008] As described above, while conventional methods can produce non-diapause eggs from diapause strains of silkworms, they suffer from the problem of a significant drop in hatching rate due to the subsequent microinjection process. Because genetic recombination occurs only in a portion of the microinjected eggs, a low hatching rate after microinjection inevitably reduces the success rate of producing genetically modified silkworms. Therefore, in order to efficiently produce genetically modified silkworms, a technology was needed that could not only obtain non-diapause eggs but also maintain the hatching rate after microinjection.

[0009] To solve the above problems, Patent Document 2 discloses a method of microinjecting a diapause hormone antibody into non-diapause eggs obtained from a diapause strain of silkworms. It is disclosed that this method increases the hatching rate after microinjection, regardless of the silkworm strain. However, this method is also labor-intensive, requiring two injections in addition to the preparation of the antibody.

[0010] Non-Patent Document 6 discloses a method for microinjection into eggs that have been treated with corona discharge to break diapause. This method allows treatment in a gaseous atmosphere and shortens the corona discharge treatment time. Furthermore, it can achieve a high hatchability rate of over 95%, making it an extremely advantageous method. However, this method also has problems. As mentioned above, if microinjection is not performed on eggs 2 to 8 hours after oviposition, when embryo development is progressing, the subsequent emergence rate of transformants is significantly reduced. Therefore, diapause-breaking treatment must be performed within the limited time between oviposition and microinjection. However, with conventional corona discharge devices, the electric field discharge range is extremely localized, making it impossible to apply it to a large number of eggs in a single treatment. As a result, the number of eggs that can be broken from diapause within a limited time is limited, resulting in poor production efficiency of transgenic silkworms.

[0011] Furthermore, genetically modified silkworms obtained by conventional microinjection have the problem that the genome composition of each transformant varies, even if the target gene introduced is the same. This is because when genetic modification is performed using the transferase activity of a transposon, the insertion of the target gene occurs at a random position on the genome, and because silkworm strains are maintained as genetically heterogeneous populations through crossbreeding.

[0012] In addition, silkworms are generally stored in the form of eggs, which can only be stored for a maximum of one year. Therefore, to maintain a strain, they must be reared and bred every year to obtain the next generation of eggs. Repeated breeding over a long period of time makes it difficult to ensure genetic stability even within the same strain.

[0013] Therefore, with conventional techniques, the expression level and quality of the target gene differ between strains or individuals of the transformant, resulting in the problem of inconsistent quality and production of the protein produced, which is a major problem when using a mass production system using genetically modified silkworms to produce raw materials for human pharmaceuticals, which require high quality.

[0014] As described above, the production of transgenic silkworms and the maintenance of their lineages require a great deal of time and effort, which increases costs. Furthermore, even if time and effort are spent, the transgenic silkworms obtained are still genetically unstable.

[0015] Therefore, new technologies are needed that can reduce costs by producing transgenic silkworms in a short time and with minimal labor, and at the same time, technologies are also needed to maintain the genetic stability of the transgenic silkworms that have been produced. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] Japanese Patent Application Laid-Open No. 2003-88274 [Patent Document 2] Patent No. 6765803 [Non-patent literature]

[0017] [Non-Patent Document 1] Tamura T., et al. (2000) Nat. Biotechnol.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. [Non-patent document 3] Zhao AC, et al., 2012, Insect Science, 19: 172-182 [Non-patent document 4] Eiichi Ozegawa et al., 2000, Japanese Journal of Sericulture, 69(6): 369-375 [Non-Patent Document 5] Shimizu, Isamu, 1991, Odokon, 35: 81-91 [Non-patent document 6] Zhang Yu-Li, et al., 2022, Frontiers in Bioengineering and Biotechnology, 10, Article853543 Summary of the Invention [Problem to be solved by the invention]

[0018] The objective of this project is to develop and provide a technology for producing practical strains of genetically modified silkworms suitable for industrial use in a relatively short period of time with little labor, and to develop and provide a technology for maintaining the genetic stability of the produced genetically modified silkworms, thereby reducing the costs required for producing genetically modified silkworms and maintaining the strains. [Means for solving the problem]

[0019] To solve the above problems, the inventors conducted extensive research and came up with a method that is the exact opposite of conventional methods: microinjecting diapause eggs and then breaking diapause by treating them with non-thermal plasma. In the technical field, when producing transgenic silkworms using diapause eggs, it is common knowledge to perform microinjection after breaking diapause, and the idea of ​​breaking diapause after microinjection is unheard of. However, with this method, eggs are microinjected 2 to 8 hours after oviposition and then diapause-breaking treatment is performed, allowing for the processing of a large number of eggs without the time constraints of conventional methods. Furthermore, it was found that the hatching rate of eggs obtained using this method was similar to that of eggs that underwent nucleic acid introduction after diapause breaking, but the positive rate of transformants was more than double that of eggs obtained using this method.

[0020] The present invention is based on this new finding and provides the following. (1) A method for producing genetically modified silkworms, comprising: a fertilized egg collection step for collecting fertilized eggs from individuals of a diapausing silkworm strain; a nucleic acid introduction step for introducing a target nucleic acid into the fertilized eggs by microinjection; a diapause-breaking step for breaking the diapause of the fertilized eggs by non-thermal equilibrium plasma treatment; and a recombinant selection step for selecting genetically modified silkworms from the next generation silkworms hatched from the fertilized eggs. (2) The method according to (1), wherein the step of breaking dormancy is carried out after the step of introducing nucleic acid. (3) The method according to (1) or (2), wherein the nucleic acid introduction step is carried out within 8 hours after collection of the fertilized egg. (4) A method for producing genetically modified silkworms, comprising: an unfertilized egg collection step of collecting unfertilized eggs from individuals of a parthenogenetic silkworm strain; a parthenogenetic induction step of subjecting the unfertilized eggs collected in the unfertilized egg collection step to a parthenogenetic induction treatment; a nucleic acid introduction step of introducing a target nucleic acid into the unfertilized eggs by microinjection; a diapause-breaking step of breaking the diapause of the unfertilized eggs by non-thermal equilibrium plasma treatment; and a recombinant selection step of selecting genetically modified silkworms from the next generation silkworms hatched from the unfertilized eggs. (5) The method according to (4), wherein the step of breaking dormancy is carried out after the step of introducing nucleic acid. (6) The method according to (4) or (5), wherein the parthenogenesis induction treatment is a high-temperature treatment in which the unfertilized eggs are exposed to 45°C to 50°C for 15 to 20 minutes. (7) The method according to any one of (4) to (6), wherein the nucleic acid introduction step is carried out within 24 hours after parthenogenesis induction treatment. (8) The method according to any one of (1) to (7), wherein the non-thermal equilibrium plasma is generated by corona discharge or dielectric barrier discharge. (9) The method according to (8), wherein the voltage in the corona discharge is 1 kV to 50 kV. (10) The method according to any one of (1) to (9), wherein a marker gene is further introduced in the nucleic acid introduction step. (11) The method according to (10), wherein the selection in the recombinant selection step is based on the expression of the marker gene. [Effects of the Invention]

[0021] According to the method for producing genetically modified silkworms of the present invention, a technology can be provided for producing practical strains of genetically modified silkworms or genetically modified cloned silkworms suitable for industrial use in a relatively short period of time with little effort. [Brief explanation of the drawings]

[0022] [Figure 1] This is a flow diagram for producing genetically modified silkworms using fertilized eggs of the present invention. This diagram illustrates a flow in which a fertilized egg collection step (S0101) is followed by a nucleic acid introduction step (S0102) and then a diapause-breaking step (S0103). However, the order of the nucleic acid introduction step (S0102) and the diapause-breaking step (S0103) can be arbitrary, and the nucleic acid introduction step (S0102) may be performed after the diapause-breaking step (S0103). [Figure 2] This is a flow diagram for producing genetically modified silkworms using unfertilized eggs of the present invention. This diagram illustrates a flow in which the parthenogenesis induction step (S0106) is followed by the nucleic acid introduction step (S0102), and then the diapause-breaking step (S0103). However, the nucleic acid introduction step (S0102) and the diapause-breaking step (S0103) can be performed in any order, and the nucleic acid introduction step (S0102) may be performed after the diapause-breaking step (S0103). [Figure 3] 1 is a conceptual diagram of a discharge device used in non-thermal equilibrium plasma treatment generated by corona discharge in Example 1. FIG. [Figure 4] FIG. 1 is a conceptual diagram of a plasma device used for non-thermal equilibrium plasma processing generated by dielectric barrier discharge in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0023] 1. Method for producing genetically modified silkworms Overview The present invention relates to a method for producing transgenic silkworms. The method is characterized in that it involves introducing a nucleic acid of interest into diapause eggs and breaking the diapause by non-thermal plasma treatment, thereby preventing the diapause of the diapause eggs or awakening them from the diapause state. According to the present invention, transgenic silkworms or transgenic cloned silkworms of practical strains suitable for industrial use can be produced in a relatively short period of time and with little effort.

[0024] 1-2.Definition As used herein, the following terms are defined. "Genetic recombination" refers to the artificial modification of the natural genetic information possessed by a host organism. Modification of genetic information here includes addition, deletion, substitution, etc. of genetic information. Artificial modification of genetic information can be achieved by existing genetic recombination techniques. For example, methods include using vectors such as plasmids or transposons to add genetic information not possessed by the host organism, or methods to destroy genetic information possessed by the host organism. Furthermore, in this specification, genome editing techniques that modify the genomic information of a host organism by genome editing are also encompassed as genetic recombination in a broad sense.

[0025] "Genetically modified silkworms" (transgenic silkworms) refer to genetically modified silkworms or their progeny produced using genetic engineering techniques. As used herein, genetically modified silkworms refer to, but are not limited to, genetically modified silkworms obtained by introducing foreign DNA into silkworm eggs by microinjection.

[0026] The term "cloned silkworms" refers to silkworm individuals that have the same genome composition and constitute a genetically homogeneous population with the same gene composition. Generally, a silkworm population obtained by inducing parthenogenesis using unfertilized eggs obtained from a parthenogenetic strain of silkworms, as described below, corresponds to cloned silkworms.

[0027] As used herein, "genetically modified cloned silkworms" refers to genetically modified cloned silkworms. In genetically modified cloned silkworms, the introduced foreign DNA is identical among individuals belonging to a clone population. Therefore, the quality and expression level of the protein encoded by the foreign DNA produced by each individual genetically modified cloned silkworm are, in principle, identical.

[0028] As used herein, a "line" refers to a population of individuals sharing specific genetic traits within the same species, and is roughly the same concept as a "strain." In this specification, lineages also encompass mutants with specific gene mutations and varieties with common morphology or properties. In principle, the biological species covered in this specification is the silkworm. Therefore, unless otherwise specified, lineages in this specification refer to "silkworm lineages," and silkworm individuals belonging to each lineage are referred to as "lineage silkworms." For example, the dormant lineages described below are dormant lineages of silkworms, i.e., "dormant silkworm lineages," and silkworms belonging to these dormant silkworm lineages are referred to as "dormant lineage silkworms." Note that when focusing on different genetic traits, a single individual may belong to multiple lineages. For example, a dormant parthenogenetic lineage has two genetic traits, dormancy and parthenogenesis. Therefore, when focusing on dormancy, it belongs to a dormant lineage, and when focusing on parthenogenesis, it belongs to a parthenogenetic lineage.

[0029] As used herein, the term "dormancy (state)" refers to a state in which an organism temporarily ceases development, growth, or activity at a specific time in its life cycle and goes into a dormant state.

[0030] As used herein, "avoiding diapause" means that diapause eggs, which have the potential to naturally enter a diapause state, continue to develop or are able to continue to develop without entering a diapause state through artificial treatment such as breaking the diapause described below.

[0031] As used herein, "awakening from diapause" refers to the awakening of diapause eggs from the diapause state by artificial treatment such as diapause-breaking, as described below, and the resumption of development.

[0032] As used herein, "diapause (silkworm) strain" refers to a population of individuals that lay diapause eggs due to the specific genetic trait of egg diapause. Silkworms are classified into univoltine, bivoltine, and multivoltine strains. Of these, many univoltine strains are diapause silkworm strains that lay diapause eggs. A "univoltine strain" is a strain that produces adults once a year when reared under natural conditions, a "bivoltine strain" is a strain that produces adults twice a year, and a "multivoltine strain" is a strain that produces adults multiple times a year. Generally, many derived strains that have an ancestral strain in temperate regions with winter are univoltine strains. However, even diapause silkworm strains can be adapted to lay non-diapause eggs by the non-diapause egg-laying treatment described below. Specific examples of dormant silkworm strains include, but are not limited to, Daizo, Nihon No. 137, Shi No. 146, Nihon No. 603, Nihon No. 604, Naka No. 604, Naka No. 605, Naka No. 514, Naka No. 515, Naka No. 9.0, Nihon No. 9.0, Shunrei, Kanetsuki, Hitachi, Nishiki, Nihon No. 502, Shi No. 146, Shi No. 122, Tokushi No. 2, Ou No. 7, Tokushi No. 4, and Kakushina.

[0033] As used herein, "diapause eggs" refer to eggs obtained from diapause-prone silkworms that have the ability to transition to diapause. Whether or not they actually transition to diapause is irrelevant. Diapause eggs can typically be obtained by oviposition by adult female diapause-prone silkworms under standard rearing conditions. Diapause eggs cease embryonic development at the embryonic stage approximately two days after oviposition and enter a cold-tolerant diapause state (Yanaginuma Toshinobu, 2015, Sericulture and Insect Biotech, 84: 100). This is thought to be a life cycle control phenomenon based on environmental responses acquired by silkworms to survive the year. Therefore, many derived strains with ancestral strains in temperate regions where winter is present are generally diapause-prone.

[0034] As used herein, "non-diapause (silkworm) strain" refers to a population of individuals that lay non-diapause eggs that do not enter a diapause state during the egg-laying stage. Generally, many of the derived strains that have ancestral strains in subtropical or tropical regions where there is no winter are multivoltine silkworm strains that repeat multiple generations per year, and these are non-diapause silkworm strains that lay non-diapause eggs. Specific examples of non-diapause silkworm strains include, but are not limited to, Mysore, Nistari, Pure Mysore, Annan, and Wagetsu.

[0035] As used herein, "breaking diapause" refers to subjecting diapause eggs to various artificial treatments to prevent them from transitioning to a diapause state or to awakening diapause eggs that are in a diapause state. As mentioned above, diapause eggs laid by diapause-prone silkworms usually enter a diapause state, with early embryonic development halting at the embryonic stage around two days after egg laying. However, by breaking diapause, diapause eggs do not enter a diapause state and continue to develop. Furthermore, if diapause eggs have already entered a diapause state and embryonic development has been stopped, diapause-breaking, such as by non-equilibrium plasma treatment, will awaken them from the diapause state and resume the stopped embryonic development.

[0036] As used herein, "non-diapause eggs" refer to silkworm eggs that do not have the property of entering a diapause state. Examples include non-diapause eggs obtained from multivoltine strains, non-diapause eggs obtained from diapause silkworm strains by non-diapause egg-laying treatment, and non-diapause eggs obtained by the method described in Japanese Patent No. 6765803. Note that diapause eggs that have avoided diapause by breaking diapause are distinguished from non-diapause eggs in this specification because they have the property of entering a diapause state.

[0037] As used herein, the term "non-diapause egg-laying treatment" refers to a specific treatment that is performed on individuals of a diapause silkworm strain that normally lays diapause eggs, thereby causing them to lay non-diapause eggs. A specific example of the specific treatment is the low-temperature dark incubation treatment described in JP 2017-085958 A.

[0038] As used herein, "parthenogenetic (silkworm) line" refers to a line in which parthenogenesis is induced with high efficiency. In parthenogenetic silkworm lines, parthenogenesis is induced by applying physical or chemical stimuli to unfertilized eggs. Generally, most silkworm lines capable of parthenogenesis are dormant silkworm lines. Therefore, in this specification, parthenogenetic silkworm lines are also referred to as "dormant parthenogenetic (silkworm) lines." Parthenogenetic silkworm lines are not limited to, but include, for example, the PK1 line, the P14 line, and a hybrid of Camboge x Nihon 106.

[0039] A "marker gene" is a polynucleotide consisting of a base sequence that encodes a marker protein, also called a selectable marker.

[0040] A "labeled protein" refers to a protein that can confer a new trait not present in the host silkworm upon expression of a marker gene. The type of labeled protein is not particularly limited, as long as its activity can be detected by a method known in the art. Preferably, the labeled protein is one that is less invasive to the transformant upon detection. Examples include fluorescent proteins, pigment-synthesizing proteins, luminescent proteins, exocrine proteins, and proteins that control external morphology. Fluorescent proteins, pigment-synthesizing proteins, luminescent proteins, and exocrine proteins are particularly suitable because they can be visually detected under specific conditions without changing the external morphology of the transformant, are very less invasive to the transformant, and allow for easy identification and selection of transformants.

[0041] As used herein, the term "fluorescent protein" refers to a protein that emits fluorescence of a specific wavelength when irradiated with excitation light of a specific wavelength. It may be either natural or non-natural. There are no particular limitations on the excitation wavelength or fluorescence wavelength. Specific examples include CFP, RFP, DsRed (including derivatives such as 3xP3-DsRed), YFP, PE, PerCP, APC, GFP (including derivatives such as EGFP and 3xP3-EGFP), etc.

[0042] As used herein, a "pigment synthesis protein" refers to a protein, typically an enzyme, involved in the biosynthesis of a pigment. The term "pigment" as used herein refers to a low-molecular-weight compound or peptide capable of imparting a pigment to a transformant, regardless of type. Preferably, the pigment is one that appears as the external color of the individual. Examples include melanin-based pigments (including dopamine melanin), ommochrome-based pigments, and pteridine-based pigments.

[0043] As used herein, the term "photoprotein" refers to a substrate protein that can emit light without the need for excitation light or an enzyme that catalyzes the luminescence of the substrate protein. Examples of the substrate protein include luciferin or aequorin, and the enzyme luciferase.

[0044] As used herein, the term "exocrine protein" refers to a protein secreted outside a cell or body, and includes exocrine enzymes, as well as fibrous proteins such as fibroin and sericin. Exocrine enzymes include enzymes such as blasticidin that contribute to the decomposition or inactivation of drugs and confer drug resistance to the host, as well as digestive enzymes.

[0045] In this specification, "non-thermal equilibrium plasma," also called non-equilibrium plasma or low-temperature plasma, refers to a relatively low-temperature plasma generated by gas discharge under atmospheric or low pressure. In thermal equilibrium plasma generated by gas discharge under atmospheric pressure, the electron temperature, ion temperature, and atomic temperature are in thermal equilibrium, whereas in non-thermal equilibrium plasma, only the electron temperature is high, while the ion and atomic temperatures are at room temperature. There are no particular restrictions on the method for generating non-thermal plasma. Examples include plasma generated by corona discharge, in which a high voltage is applied to a needle-to-plane electrode or other electrode system that forms a significantly non-uniform electric field, and plasma generated by dielectric barrier discharge, in which an AC voltage is applied between electrodes with a dielectric disposed therebetween.

[0046] Since the target of non-thermal plasma treatment is silkworm eggs, the temperature of the generated plasma is not limited, but it is preferable that the plasma be, for example, 35°C to 80°C, 40°C to 75°C, 45°C to 70°C, 50°C to 65°C, or 55°C to 60°C.

[0047] In this specification, the term "corona discharge" refers to a gas discharge phenomenon in which, when a voltage is applied to a needle-to-plane electrode or other electrode system that forms a significantly non-uniform electric field, local dielectric breakdown occurs in the gas between the electrodes, causing a current to flow.

[0048] As used herein, the term "dielectric barrier discharge" refers to a gas discharge phenomenon that occurs in a gas between electrodes when a voltage is applied between electrodes with a dielectric disposed therebetween. The "voltage" referred to here is not limited. For example, it includes voltages from commercial AC power sources, high-frequency or microwave power sources, pulse power sources, and other power sources.

[0049] 1-3.Production method The flow chart of the method for producing transgenic silkworms of the present invention is shown in Figure 1. As shown in this figure, the production method of the present invention includes a fertilized egg collection step (S0101), a nucleic acid introduction step (S0102), a diapause-breaking step (S0103), a recombinant selection step (S0104), an unfertilized egg collection step (S0105), and a parthenogenesis induction step (S0106).

[0050] The method for producing genetically modified silkworms of the present invention can be broadly divided into a method for producing genetically modified silkworms using fertilized eggs and a method for producing genetically modified silkworms using unfertilized eggs.

[0051] Of the above steps, the four steps of the fertilized egg collection step (S0101), the nucleic acid introduction step (S0102), the diapause-breaking step (S0103), and the recombinant selection step (S0104) are steps used in the method for producing genetically modified silkworms using fertilized eggs, and the five steps of the nucleic acid introduction step (S0102), the diapause-breaking step (S0103), the recombinant selection step (S0104), the unfertilized egg collection step (S0105), and the parthenogenesis induction step (S0106) are steps used in the method for producing genetically modified silkworms using unfertilized eggs. Note that in both the method for producing genetically modified silkworms using fertilized eggs and the method for producing genetically modified silkworms using unfertilized eggs, the order of the nucleic acid introduction step (S0102) and the diapause-breaking step (S0103) does not matter, and either may be performed first. Each step in the method for producing a transgenic silkworm of the present invention will be specifically described below.

[0052] 1-3-1. Fertilized egg collection process The "fertilized egg collection step" (S0101) is a step specific to the method for producing genetically modified silkworms using fertilized eggs, and is a selection step for collecting dormant fertilized eggs to be used in the nucleic acid introduction step (S0102). This step is performed to obtain fertilized eggs of G0 silkworms (nucleic acid introduction generation) and fertilized eggs of the G1 silkworms (first generation after nucleic acid introduction). The genetically modified silkworms obtained in this case are normal genetically modified silkworms, not cloned silkworms. On the other hand, this step may be performed only on fertilized eggs of G0 silkworms (nucleic acid introduction generation), and unfertilized eggs may be obtained from the G1 silkworms in the unfertilized egg collection step (S0105) described below. The genetically modified silkworms obtained in this case are genetically modified cloned silkworms.

[0053] Diapause fertilized eggs can be prepared by allowing mated female adult individuals to lay eggs naturally according to a known egg collection method. The female individuals used for egg collection can be a diapause silkworm strain that lays diapause eggs. Eggs laid by mated female adult individuals from a diapause silkworm strain are, in principle, diapause fertilized eggs.

[0054] There are no limitations on the egg collection method. Usually, a mated adult female is provided with an egg-laying mat, and the adult female is allowed to lay eggs on the mat. When a mated adult female is provided with an egg-laying mat, egg-laying often begins within a few hours of the mating. After this step, dormant fertilized eggs can be obtained for use in the next nucleic acid transfer step.

[0055] 1-3-2. Nucleic acid introduction step The "nucleic acid introduction step" (S0102) is a step of introducing a target nucleic acid into an egg. The eggs used in this step are fertilized or unfertilized eggs of G0 silkworms. In this step, it is desirable to introduce the nucleic acid into multiple eggs.

[0056] In this process, the "nucleic acid of interest" refers to a nucleic acid to be introduced into silkworms to produce the desired transgenic silkworms. Examples include DNA and RNA. More specific examples include, but are not limited to, donor DNA / RNA, helper DNA / RNA, or guide DNA / RNA. Preferably, the nucleic acid promotes incorporation into the genome, such as a gene of interest, an expression vector containing the gene, or a helper plasmid. When introducing a gene, the type of gene is not important. For example, a gene encoding a protein or a functional nucleic acid (such as an RNAi molecule) may be used. A desired gene capable of conferring a desired trait can be introduced. A marker gene may also be introduced to facilitate the selection of individuals into which the nucleic acid of interest has been introduced in the selection process described below. In this process, if necessary, a peptide (including proteins such as enzymes) and / or a low-molecular-weight compound can also be introduced together with the nucleic acid. In this case, the order of introduction with the nucleic acid is not important. The introduction may be before, after, or simultaneously with the nucleic acid introduction.

[0057] The gene introduced in this step can be of any origin. It may be derived from the host silkworm, or from another organism, such as a human. For example, when producing an antibody using a transgenic silkworm, the foreign DNA can be an expression unit containing an IgG antibody gene and gene expression regulatory regions such as a promoter and a terminator.

[0058] This step can be carried out by methods known in the art for introducing foreign genes into silkworms. For example, if the expression vector is a plasmid containing transposon inverted terminal repeats at both ends of the target DNA (Handler AM. et al., 1998, Proc. Natl. Acad. Sci. USA 95:7520-5), the method of Tamura et al. (Tamura T. et al., 2000, Nature Biotechnology, 18, 81-84) or the method of Zhou et al. (Zhou W. et al., 2012, Insect Science, 19:172-182) can be used. Specifically, the expression vector is dissolved or diluted with a solvent such as water or buffer to an appropriate concentration to prepare an injection solution. A helper plasmid containing DNA encoding a transposon transferase is added to the injection solution. An example of the helper plasmid is pHA3PIG.

[0059] Although not limited to, the introduction of a nucleic acid of interest into an egg is generally carried out using a special injection device that utilizes air pressure. For example, the method described in Japanese Patent No. 1654050 or the method of Tamura et al. (Tamura T, et al., 2007, J. Insect. Biotechnol. Sericol., 76: 155-159) may be used. The amount of nucleic acid to be introduced is not particularly limited and may be determined appropriately depending on the type, properties, and purpose of the nucleic acid. The amount is usually 1 nL to 5 nL.

[0060] 1-3-3. Dormant breaking process The "diapause-breaking step" (S0103) is an essential step common to the methods for producing genetically modified silkworms using either fertilized eggs or unfertilized eggs, and is a step in which diapause-breaking treatment is performed on diapause eggs. Diapause eggs are artificially treated to break diapause, thereby preparing diapause eggs that have avoided diapause or diapause-awakened eggs.

[0061] The resting eggs used in this step may be either fertilized or unfertilized. However, in the case of unfertilized eggs, they are eggs derived from a parthenogenetic silkworm strain and are resting eggs that have undergone the unfertilized egg collection step (S0105) and parthenogenesis induction step (S0106) described below. Furthermore, the resting eggs used may be not only fertilized or unfertilized eggs of G0 silkworms, but also fertilized or unfertilized eggs of G1 silkworms.

[0062] As mentioned above, diapause eggs transition to a diapause state two or more days after oviposition. Therefore, when using diapause eggs before they enter a diapause state in this step, it is desirable to perform this step on eggs within 1 to 50 hours, 2 to 48 hours, 3 to 42 hours, 4 to 36 hours, 6 to 30 hours, or 8 to 24 hours after oviposition. Furthermore, as described in the unfertilized egg collection step (S0105) described below, when eggs from a parthenogenetic silkworm strain are used, unfertilized eggs can be collected directly from ovaries removed by dissection or other methods without oviposition. When eggs are protected at, for example, 15°C after parthenogenesis induction, they transition to a diapause state after six days or more. Therefore, it is desirable to perform this step on eggs less than 144 hours (6 days), within 126 hours, within 10 hours, within 90 hours, or within 72 hours after parthenogenesis induction.

[0063] On the other hand, when using diapause eggs that have entered a diapause state in this process, it is desirable to carry out the process on eggs that are 36 to 158 hours, 48 ​​to 144 hours, 60 to 132 hours, 72 to 120 hours, or 84 to 96 hours after laying.

[0064] Unless otherwise specified, the diapause-breaking treatment performed in this step is a non-thermal equilibrium plasma treatment. As used herein, "non-thermal equilibrium plasma treatment" refers to exposing diapause silkworm eggs to non-thermal equilibrium plasma under atmospheric or low pressure. This is a method for preventing diapause eggs from transitioning to a diapause state or awakening them from the diapause state. Non-thermal plasma treatment includes, but is not limited to, treatment with plasma generated by corona discharge (corona discharge plasma) or plasma generated by dielectric barrier discharge (dielectric barrier discharge plasma).

[0065] The voltage in the corona discharge or dielectric barrier discharge is not limited, but is preferably 1 kV or more, 2 kV or more, 3 kV or more, 4 kV or more, 5 kV or more, 6 kV or more, 7 kV or more, 8 kV or more, 9 kV or more, 10 kV or more, 12 kV or more, 13 kV or more, or 15 kV or more, and 50 kV or less, 45 kV or less, 40 kV or less, 35 kV or less, 30 kV or less, 25 kV or less, or 20 kV or less. The current is not limited, but may be in the range of 0.05 mA to 0.5 mA, 0.1 mA to 0.45 mA, 0.15 mA to 0.4 mA, 0.2 mA to 0.35 mA, or 0.25 mA to 0.3 mA. The discharge time is not limited, but may be within the range of 0.1 seconds to 20 minutes, 0.5 seconds to 18 minutes, 1 second to 15 minutes, 5 seconds to 14 minutes, 10 seconds to 12 minutes, 30 seconds to 10 minutes, 45 seconds to 8 minutes, 1 minute to 6 minutes, 1 minute 30 seconds to 5 minutes, 2 minutes to 4 minutes, or 2 minutes 30 seconds to 3 minutes.

[0066] The non-thermal equilibrium plasma treatment can be performed on diapausing eggs in the atmosphere. If necessary, the diapausing eggs can be fixed to a substrate such as paper with an adhesive.

[0067] This step prevents diapause in the diapause eggs or causes them to break out of diapause. After breaking diapause, the eggs can be incubated under appropriate conditions, for example, at 25°C, until they hatch.

[0068] 1-3-4. Recombinant selection process The "recombinant selection step" (S0104) is a step of selecting genetically modified silkworms from silkworms that have hatched from eggs that have been subjected to the nucleic acid introduction step (S0102) and diapause-breaking step (S0103).

[0069] G0 silkworms that have undergone the diapause-breaking step (S0103) contain genetically modified somatic cells and germ cells, but it is the genetic modifications that have occurred in the germ cells that are passed on to the next generation. Therefore, this step is carried out on G1 silkworms.

[0070] As shown in Figure 1, when G1 fertilized eggs obtained through the fertilized egg collection step (S0101) from G0 silkworms of a diapause-breaking silkworm strain that have been subjected to non-thermal plasma treatment in the diapause-breaking step (S0103) following the nucleic acid introduction step (S0102) are used in this step (S0104), the diapause-breaking step (S0103) is again performed on the G1 fertilized eggs before this step is carried out. The silkworms obtained after this step are conventional genetically modified silkworms. Since no gene is introduced into the G1 fertilized eggs by microinjection, the risk to the eggs can be reduced. Therefore, the diapause-breaking treatment for the G1 fertilized eggs is not limited to non-thermal plasma treatment, and other known diapause-breaking methods can also be used. Examples include DMSO treatment, acid immersion treatment, centrifugation, and oxygen treatment.

[0071] As shown in Figure 2, when G1 unfertilized eggs obtained from G0 silkworms of a parthenogenetic silkworm strain that have been treated with non-thermal plasma in the diapause-breaking step (S0103) following the nucleic acid introduction step (S0102) are used, a parthenogenesis induction step (S0106) is performed on the G1 unfertilized eggs before this step (S0104), and then a diapause-breaking step (S0103) is performed on the individuals that hatch after this diapause-breaking step (S0103). Therefore, the silkworm individuals obtained after this step are each individual first-generation genetically modified cloned silkworms (G1).

[0072] Furthermore, in exceptional cases, G0 silkworms developed from G0 fertilized eggs collected by crossbreeding or the like may contain G1 unfertilized eggs, which are expected to have a high rate of parthenogenesis. In such cases, as shown in Figure 1, even if the G0 silkworms have undergone the fertilized egg collection step (S0101), an unfertilized egg collection step (S0105) can be performed, in which unfertilized eggs are collected from G0 female individuals after the diapause-breaking step (S0103). The obtained G1 unfertilized eggs can then be subjected to the parthenogenesis induction step (S0106) and, if necessary, the diapause-breaking step (S0103), before this step is carried out. In this case, the silkworm individuals obtained after this step will each be an individual first-generation (G1) genetically modified cloned silkworm.

[0073] Selection of genetically modified organisms may be performed by methods known in the art. For example, selection may be performed based on the traits brought about by the nucleic acid of interest introduced in the nucleic acid introduction step (S0102). For example, if the nucleic acid of interest is a foreign gene, genomic DNA or mRNA may be prepared from silkworms that have emerged after the nucleic acid introduction step (S0102), and the presence or absence of the foreign gene or the expression of the foreign gene may be confirmed by PCR or the like.

[0074] Alternatively, if the expression vector used for nucleic acid introduction contains a marker gene, the desired transgenic silkworms can be easily selected based on the expression of the marker gene. The marker protein produced by the expression of the marker gene can confer new traits not possessed by the host silkworm. Based on the activity of this marker protein, transformants carrying the introduced nucleic acid of interest can be easily identified. Here, "based on activity" means based on the results of activity detection. Activity detection may be performed by directly detecting the activity of the marker protein itself, or indirectly via a metabolite, such as a dye, generated by the activity of the marker protein. Detection may be chemical (including enzyme reaction detection), physical (including behavioral analysis detection), or sensory detection by the detector (including detection by sight, touch, smell, hearing, or taste).

[0075] 1-3-5.Unfertilized egg collection process The "unfertilized egg collection step" (S0105) is, in principle, a step of collecting unfertilized eggs from individuals of a parthenogenetic silkworm strain. This step is carried out together with the parthenogenetic induction step (S0106) described below when a parthenogenetic silkworm strain is used as the parent strain to produce a genetically modified cloned silkworm. This step is essential when producing a genetically modified cloned silkworm from a parthenogenetic silkworm strain.

[0076] Furthermore, this process can be carried out exceptionally even if the G0 silkworms obtained through the fertilized egg collection process are not a parthenogenetic silkworm strain, provided that the G1 eggs obtained from the G0 silkworms can develop with a high probability through parthenogenetic induction treatment.

[0077] Therefore, when performing this step and the parthenogenesis induction step (S0106) described below, the dormant eggs used in the nucleic acid introduction step (S0102) are, in principle, dormant unfertilized eggs obtained from individuals of a parthenogenetic silkworm strain, but as an exception, as mentioned above, they may also be dormant unfertilized eggs or non-dormant unfertilized eggs obtained from individuals that have undergone the fertilized egg collection step.

[0078] In this step, although there are no particular limitations, it is preferable to obtain from adult female individuals unfertilized eggs that are capable of parthenogenesis in vivo, that is, unfertilized eggs that have developed into mature unfertilized eggs.

[0079] The method for collecting unfertilized eggs is not limited, and examples thereof include collection by dissection and collection by natural spawning.

[0080] Specifically, collection by dissection involves, for example, cutting open the abdomen or tail of a female adult with a scalpel or dissecting scissors, and then applying pressure to the abdomen with your fingers to remove the ovaries by pushing them out through the incision. The removed ovaries can be immersed in water to loosen the clumped oviducts. Next, the ovaries are placed on a fine-mesh stainless steel mesh or gauze, and rubbed with a finger or other device to separate the oviduct tissue from the unfertilized eggs. Finally, water is poured through the mesh to remove the floating oviduct tissue, and the unfertilized eggs remaining at the bottom are collected. Repeating this procedure several times allows only the unfertilized eggs to be collected.

[0081] Unfertilized eggs can also be collected by natural egg-laying, in which virgin female individuals are allowed to lay eggs. The egg-collection method is not limited. For example, a method is employed in which a female individual is provided with an egg-laying mat after hatching and allowed to lay eggs on the mat. Specifically, the female individual is placed at a low temperature of 0 to 10°C, preferably 5°C, after hatching, and stored at that temperature for 1 to 2 days, and at most 1 to 7 days. The female silkworm is then transferred from the low temperature to room temperature (23 to 28°C), where it is provided with an egg-laying mat and allowed to start laying eggs in the dark.

[0082] 1-3-6. Parthenogenesis induction process The "parthenogenesis induction step" (S0106) is a step of inducing parthenogenesis in the unfertilized eggs obtained in the unfertilized egg collection step (S0105). This step is carried out in conjunction with the unfertilized egg collection step (S0105).

[0083] The parthenogenetic induction treatment may be carried out according to a method known in the art and is not particularly limited. For example, the induction methods disclosed in Sugai et al. (Etsuji Sugai et al., 1983, Journal of Japanese Sericultural Science, Vol. 52, No. 1: 51-56), Hirokawa (Masahiko Hirokawa, 1990, Fukushima Sericultural Research Institute Bulletin, 24: 1-6), and Kosegawa et al. (Kosegawa E., et al., 2012, J. Insect. Biotechnol. Sericology, 81: 37-44) may be referenced. Parthenogenetic induction is generally induced by applying physical or chemical stimuli to unfertilized eggs. Specific examples include high-temperature treatment. A specific example of high-temperature treatment is to expose the unfertilized eggs collected in the above step to hot water at 45°C to 50°C or 46°C to 48°C for 15 to 20 minutes or 16 to 18 minutes. Thereafter, it is preferable to keep the mixture at 12°C to 18°C, 13°C to 17°C, 14°C to 16°C, or 15°C for 2 to 6 days, or 3 to 5 days.

[0084] The dormant unfertilized eggs obtained after this process have been induced to parthenogenetically develop. However, because they are dormant eggs, they will not hatch. By subjecting these dormant eggs to the diapause-breaking process (S0103) described above, diapause can be avoided and development will continue, leading to the eggs eventually hatching. [Example]

[0085] <Example 1: Production of transgenic silkworms using diapause eggs> (the purpose) We will verify hatching after microinjection and the acquisition of genetically modified silkworms using the method for producing genetically modified silkworms of the present invention. (Methods and Results) The diapause silkworm strain "Ariake" was used in each experimental group. This strain was obtained from the National Agriculture and Food Research Organization (NARO), a national research and development agency in Japan. After obtaining the silkworms, they were reared on an artificial diet (Nippon Nosan Co., Ltd.) at 28°C until the third instar, and then reared at 25°C under a 12-hour light / 12-hour dark cycle.

[0086] 1. Verification of hatching rate by non-thermal plasma treatment In this example, corona discharge was used for non-thermal equilibrium plasma treatment. The corona discharge treatment device shown in Figure 3 was used. This treatment device consists of two components: a corona discharge device and a high-voltage current power supply (HAR-30P2, Matsusada Precision Co., Ltd.). The corona discharge device further comprises an aluminum electrode plate and four stainless steel electrode needles. The distance between the tip of the positively charged electrode needle and the negatively charged plate was adjusted within a range of 10 to 30 mm. To investigate the optimal corona discharge conditions, eggs were placed on the aluminum electrode plate with the laying paper still attached 2, 4, and 24 hours after oviposition. Corona discharge was then applied to the eggs at various intensities. This exposed the eggs to corona discharge plasma, breaking their diapause. After treatment, the eggs were transferred to a humidified plastic box and stored at 25°C for at least 14 days. The hatchability was then measured. Eggs not treated with corona discharge plasma served as a control. The results are shown in Table 1.

[0087] [Table 1]

[0088] The results in Table 1 demonstrate that non-thermal plasma treatment of diapause eggs can break diapause with a hatch rate of over 80% for eggs 2 to 24 hours after laying. When a high voltage of over 15 kV was applied, it was revealed that there was no significant difference in the effectiveness of short-term and long-term treatment in breaking diapause in diapause-breaking eggs (data not shown).

[0089] 2. Generation of Transgenic Silkworms After microinjection, diapause was broken by non-thermal plasma. Eggs laid on the surface of egg-laying paper within 2 hours of oviposition were immersed in water for 5-10 minutes to separate them from the paper, and then fixed with instant adhesive on waterproof art paper. The art paper was placed on a glass slide, and microinjection was performed. Microinjection of vector and helper plasmids into eggs was performed according to the method of Tanura et al. (Tamura T., et al. (2000) Nat. Biotechnol.18, 81-84.). The vector plasmid used was pBac[3xP3DsRedafm] (Horn and Wimmer, 2000, Dev. Genes Evol., 210(12):630-637), and the helper plasmid was pHA3PIG (Tamura T., et al., 2000, Nat. Biotechnol., 18:81-84). These plasmids were purified using the Qiagen Plasmid Hispeed Midi Kit (Qiagen). The purified plasmid DNA was dissolved in injection buffer (0.5 mM phosphate pH 7.0 / 5 mM KCl) at a concentration of 200 μg / mL and then introduced into eggs.

[0090] After microinjection, the art paper containing the eggs was removed from the slide and incubated at 25°C for 24 hours after oviposition, until the injected eggs reached the developmental stage. The eggs were then treated with corona discharge plasma at 5 kV for 5 minutes.

[0091] As a comparative example, eggs were treated with corona discharge plasma within 2 hours after oviposition (after collection of fertilized eggs), as in Zhang et al. (2022, cited above), to break diapause, and then microinjection was performed. Corona discharge was performed under the same conditions as above, on eggs laid on the surface of egg-laying paper within 2 hours after oviposition. After breaking diapause, the eggs were immersed in water for 5-10 minutes to separate them from the egg-laying paper, and then fixed to a glass slide with instant adhesive. Microinjection was performed under the above conditions on eggs within 6 hours after oviposition (collection of fertilized eggs).

[0092] The eggs were kept in a humidified plastic container in a 25°C incubator until hatching. The hatched larvae were transferred to a Petri dish and fed an artificial diet at approximately 28°C for 2–3 days. They were then transferred to a plastic container for artificial diet and reared at 25–27°C until they reached adulthood. The resulting G0 adults were either sibling-mated or backcrossed to obtain the next generation of G1 eggs. To prevent diapause in the G1 eggs, 20 hours after oviposition, the eggs were treated with 6N HCl for 1 hour at 25°C. The HCl-treated eggs were placed in a plastic container at 25°C, and the DsRed fluorescence was examined using a microscope equipped with a DsRed filter immediately before hatching. Larvae that hatched from the fluorescent eggs were reared to adulthood, establishing transgenic lines. Table 2 shows the hatching rate after injection, and Table 3 shows the calculated rate of transgenic lines (positive lines) obtained in G1.

[0093] [Table 2]

[0094] In the table, Experiment No. 1 represents the method of the present invention, in which diapause eggs were microinjected within 6 hours after oviposition, and then diapause-breaking treatment was performed when the eggs reached the developmental stage 24 hours after oviposition. On the other hand, Experiment No. 2 represents a comparative example, in which diapause-breaking treatment was performed on diapause eggs within 2 hours after oviposition, and DNA such as a vector plasmid was microinjected into the eggs within 6 hours after oviposition.

[0095] Table 2 shows that the hatching rate can be roughly the same regardless of the order of diapause-breaking treatment and microinjection.

[0096] [Table 3]

[0097] As shown in Table 3, DsRed fluorescence expression was observed in eggs in both Experiment No. 1 (G1-positive lineages 1-2) and Experiment No. 2 (G1-positive lineages 3-6). However, the frequency of positive embryos was 1.1-5.2% in Experiment No. 2, a comparative example, whereas it was 10.8-22.9% in Experiment No. 1, which was the method of the present invention, showing a positive rate more than double that of the comparative example.

[0098] The above results show that the method of the present invention can achieve a similar hatching rate and more than twice the positive rate of transgenic lines compared to the conventional method disclosed by Zhang et al. (2022, cited above), which involves microinjection after breaking diapause.

[0099] To confirm the stability of the transgenic silkworms, we raised silkworms hatched from G1 eggs to adulthood to obtain G2 eggs. We confirmed that the expression of DsRed in the eggs and adults was stable and transmitted to the next generation. We also confirmed that all established lines laid diapause eggs (data not shown).

[0100] <Example 2: Breaking dormancy by non-thermal equilibrium plasma treatment of dielectric barrier discharge> (the purpose) In Example 1, diapause of dormant eggs was broken by non-thermal plasma treatment, in which eggs were directly exposed to corona discharge applied by a DC power source. To confirm that this dormancy breaking was due to non-thermal plasma generated by corona discharge, rather than electrical stimulation from the corona discharge, we verified that non-thermal plasma treatment generated by dielectric barrier discharge could also break dormancy.

[0101] (Methods and Results) The dormant silkworm strains and rearing methods were basically the same as those in Example 1. For the dielectric barrier discharge, the treatment device shown in Figure 4 was used. This treatment device is a handy type device (HPJ-02A manufactured by Aqua Co., Ltd.) that integrates a 10-12 kV AC power supply, a high-voltage electrode for plasma generation, an earth electrode, and a nitrogen gas discharge device. By applying a high voltage between the parallel electrodes facing each other, a dielectric barrier discharge occurs between the electrodes. This discharge generates a non-thermal equilibrium plasma between the electrodes. By passing nitrogen gas discharged from the nitrogen gas discharge device between these electrodes at high speed, the nitrogen gas is converted into plasma nitrogen ions (N 3- This plasma gas was irradiated onto the surface of diapause eggs on egg-laying paper to perform non-thermal plasma treatment. In this method, the eggs are not exposed to electrical stimulation by the dielectric barrier discharge, and are treated only by the plasma gas.

[0102] To investigate the optimal conditions for plasma gas irradiation, eggs 4 and 20 hours after oviposition were irradiated with plasma gas for 10, 20, 30, 60, and 180 seconds. After treatment, the eggs were transferred to a humidified plastic box and stored at 25°C for at least 14 days to confirm whether diapause was broken by hatching. Eggs 4 and 20 hours after oviposition that had not been irradiated with plasma gas served as controls.

[0103] (result) In the experimental areas where plasma gas was irradiated, hatching was observed in all experimental areas, regardless of the time after egg laying or the duration of plasma gas irradiation, confirming the breaking of diapause in the dormant eggs. On the other hand, no hatching was observed in the control area. These results demonstrated that the breaking of diapause occurs not through electrical stimulation but through the action of non-thermal equilibrium plasma.

[0104] Example 3: Breaking diapause in parthenogenetic eggs by non-thermal plasma treatment using corona discharge (the purpose) In Example 1, the diapause of dormant fertilized eggs was broken by a non-thermal equilibrium plasma treatment generated by corona discharge applied from a DC power source. In this example, it was verified that the diapause of dormant unfertilized eggs collected from parthenogenetic female parents could be broken by this treatment.

[0105] (Methods and Results) Each experimental group used the diapausing parthenogenetic silkworm strain pK1 (obtained from the Institute of Entomology of the Academy of Sciences of the Czech Republic). After obtaining the silkworms, they were reared on an artificial diet (Nippon Nosan Co., Ltd.) at 28°C until the third instar, and then reared at 25°C under a 12-hour light and 12-hour dark cycle. The emerged female moths were then stored at 4°C, and unfertilized eggs were prepared according to the unfertilized egg collection step (S0105). Subsequently, parthenogenesis was induced in the unfertilized eggs according to the parthenogenesis induction step (S0106), and the unfertilized eggs were then stored at 15°C for 72 hours.

[0106] Corona discharge plasma treatment was performed at room temperature 0, 24, and 72 hours after parthenogenetic induction. The corona discharge plasma treatment was performed according to the method in Example 1. The corona discharge plasma conditions were 15 kV, 0.1 mA, and 5 or 10 minutes. The negative control was an untreated plant, and the positive control was a plant that had been subjected to an acid treatment 72 hours after parthenogenetic induction.

[0107] (result) The results are shown in Table 4. [Table 4]

[0108] The results in Table 4 demonstrate that corona discharge plasma treatment can break diapause in dormant unfertilized eggs collected from parthenogenetic females after parthenogenesis induction treatment, with a hatch rate of 30% to 80% for eggs 0 to 72 hours after laying. It was also revealed that when a high voltage of over 15 kV was applied, there was no significant difference in the effectiveness of diapause-breaking between short-term (5 minutes) and long-term (10 minutes) treatment, as with dormant fertilized eggs.

Claims

1. A method for producing a genetically modified silkworm, comprising: a fertilized egg collection step of collecting fertilized eggs from individuals of the diapausing silkworm strain; a nucleic acid introduction step of introducing a nucleic acid of interest into the fertilized egg by microinjection; a diapause-breaking step of breaking the diapause of the fertilized eggs after the nucleic acid introduction step by non-thermal equilibrium plasma treatment; and The production method further comprises a recombinant selection step of selecting genetically modified silkworms from next-generation silkworms hatched from the fertilized eggs.

2. The method according to claim 1, wherein the nucleic acid introduction step is carried out within 8 hours after collection of the fertilized egg.

3. A method for producing a genetically modified silkworm, comprising: an unfertilized egg collection step of collecting unfertilized eggs from individuals of the parthenogenetic silkworm strain; a parthenogenesis induction step in which the unfertilized eggs collected in the unfertilized egg collection step are subjected to parthenogenesis induction treatment; a nucleic acid introduction step of introducing a nucleic acid of interest into the unfertilized egg by microinjection; a diapause-breaking step of breaking the diapause of the unfertilized eggs after the nucleic acid introduction step by non-thermal equilibrium plasma treatment; and a step of selecting recombinants from next-generation silkworms hatched from the unfertilized eggs; The method for producing the same.

4. 4. The method according to claim 3, wherein the parthenogenetic induction treatment is a high temperature treatment in which the unfertilized eggs are exposed to 45°C to 50°C for 15 to 20 minutes.

5. The method according to claim 3 or 4, wherein the nucleic acid introduction step is carried out within 24 hours after parthenogenesis induction treatment.

6. 4. The method according to claim 1, wherein the non-thermal equilibrium plasma is generated by corona discharge or dielectric barrier discharge.

7. 7. The method according to claim 6, wherein the voltage in the corona discharge is 1 kV to 50 kV.

8. The method according to claim 1 or 3, wherein a marker gene is further introduced in the nucleic acid introduction step.

9. The method according to claim 8 , wherein the selection in the recombinant selection step is based on the expression of the marker gene.

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