Method for producing genetically engineered bombyx mori by using dormant egg

Abstract

To provide a novel method for producing genetically engineered Bombyx mori from all parent lines, and furthermore, maintaining the line in a genetically stable state.SOLUTION: A method for performing parthenogenetic induction treatment after collecting a nondormant unfertilized egg from a female individual of parthenogenetic-line Bombyx mori from which a subesophageal ganglion has been removed, and introducing an objective nucleic acid into the egg after treatment to obtain a genetically engineered clone Bombyx mori.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Silkworms (Bombyx mori) are insects that have long been used industrially to produce silk. They can mass-produce silk threads in a short period of time because they spin cocoons. This is due to the high protein production capacity of the silkworm's silk gland. Genetic engineering techniques have now made it possible to use the silkworm's capabilities to mass-produce useful proteins other than silk thread.

[0003] When silkworms are used as hosts for mass production of proteins, a technology for producing a transformant into which a foreign gene has been introduced, that is, a transgenic silkworm (transgenic silkworm), is indispensable. To produce transgenic silkworms, a microinjection method is used in which a desired gene is introduced into an egg using a transposon (Non-Patent Document 1). Silkworm eggs are fertilized within 2 hours after spawning, and then a naked nucleus without a cell membrane called a syncytium moves to the surface of the egg while repeatedly dividing. To introduce a desired gene, it is necessary to do so in an egg 2 to 8 hours after spawning, when embryo development is progressing. This is because the efficiency of emergence of transformants decreases significantly after this period (Non-Patent Document 2). In other words, to produce transgenic silkworms using existing methods, it is important to introduce a target gene into an egg in which embryo development does not stop within a specific time after spawning.

[0004] By the way, most silkworms are univoltine, in which adults usually emerge once a year. Univoltine silkworms lay diapause eggs and enter a state of diapause in the eggs. This state of diapause is maintained even after microinjection, so even if the gene is introduced into the diapause eggs, a transformant cannot be obtained. In other words, in order to carry out the microinjection method, non-diapause eggs that do not enter a state of diapause must be used, and this method has the problem of being limited to parent strains that lay non-diapause eggs. However, most of the non-diapause strains that lay non-diapause eggs are experimental strains, and have inherently poor practical qualities, so there is a further problem that they are unsuitable for industrial use. Therefore, in the past, a method was used in which genetic recombination was carried out by microinjection using non-diapause eggs obtained from a non-diapause strain, and the obtained genetically modified silkworms were then crossed with a diapause practical strain to develop into a practical strain suitable for industrial use, but this method required a great deal of effort and time.

[0005] In order to solve the above problems, methods have been developed to obtain dormant eggs or non-diapause eggs that do not enter a diapause state from practical dormant strains. For example, there are a diapause breaking method in which the transition of dormant eggs to a dormant state is prevented by external physical or chemical stimuli, thereby maintaining embryo development, and a non-diapause egg laying method in which parent silkworms of a dormant strain are treated to lay non-diapause eggs.

[0006] An example of a method for breaking diapause is the acid immersion treatment disclosed in Non-Patent Document 3. Normally, acid immersion is a method for breaking diapause by immersing diapause eggs 24 hours after spawning in a hydrochloric acid solution. In Non-Patent Document 3, bivoltine diapause eggs that were acid immersed within 3 hours after spawning are genetically modified by microinjection. However, this method has the problem that the eggs are given a physical stimulus, microinjection, in addition to the chemical stimulus of acid, which significantly reduces the subsequent hatching rate.

[0007] An example of a non-diapause egg spawning method is the low-temperature dark incubation method disclosed in Non-Patent Documents 4 and 5. The low-temperature dark incubation method involves incubating parent eggs under low-temperature, dark conditions, and allowing the emerged parent silkworms to spawn non-diapause eggs. However, the low-temperature dark incubation method has the problem that there is a significant difference in the number of non-diapause eggs laid depending on the silkworm strain, and sometimes no non-diapause eggs are laid at all (Patent Document 1). In addition, when microinjection is performed on non-diapause eggs obtained by this method, there is also the problem that the hatching rate drops drastically, as with the acid immersion method.

[0008] Therefore, Patent Document 2 discloses a diapause hormone antibody method as a technique for solving the above problems and maintaining the hatching rate after microinjection. In this method, a neutralizing antibody against the diapause hormone (anti-diapause hormone antibody) is injected into parent silkworms from the final instar larva to the early pupa stage, functionally inhibiting the diapause hormone of the female parent silkworm, thereby causing the diapause strain silkworms to lay non-diapause eggs, and microinjecting the non-diapause eggs obtained. Patent Document 2 discloses that the method increases the hatching rate after microinjection regardless of the silkworm strain. However, this method also requires the preparation of an effective neutralizing antibody and two antibody injections, which is problematic in terms of time and effort.

[0009] On the other hand, the genome composition of transgenic silkworms obtained by microinjection differs from one transformant to another even if the target gene introduced is the same. This is because when gene recombination is performed using the transferase activity derived from 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.

[0010] In addition, silkworms are generally stored in the form of eggs, which can only be stored for a maximum of one year. Therefore, in order to maintain a lineage, they must be reared and mated every year to obtain the next generation of eggs. If breeding is repeated over a long period of time, it becomes difficult to ensure genetic stability even within the same lineage.

[0011] Therefore, for the mass production of raw materials for human pharmaceuticals, which require high quality, genetically modified silkworms are needed that can provide proteins with stable quality and production volume, regardless of the strain or individual differences of the transformed organisms.

[0012] As described above, in order to develop genetically modified silkworms suitable for industrial use, it takes a huge amount of time and effort not only to create them but also to maintain a genetically stable lineage, and the resulting huge costs have been a major obstacle to their widespread practical application. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] JP 2003-88274 A [Patent Document 2] Patent No. 6765803 [Non-patent literature]

[0014] [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 on 21st Century Agriculture: Genetic Engineering Research of Animals and Microorganisms, edited by the Agricultural Society of Japan) pp57-76. 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, Ōdōkon, 35: 81-91 Summary of the Invention [Problem to be solved by the invention]

[0015] The present invention aims to develop and provide a new method for producing genetically modified silkworms from any parent line and further for maintaining the line in a genetically stable state. [Means for solving the problem]

[0016] To solve the above problems, the present inventors attempted to produce transgenic silkworms using a method for obtaining non-diapause eggs from female moths of a diapause strain by removing the subosophageal ganglion (SG), which secretes diapause hormone, rather than suppressing the function of anti-diapause hormone antibodies with neutralizing antibodies (Fukuda, S., 1952, Annot. Zool. Japan 25: 149-155). However, this method involved an unexpected problem that is essential for obtaining diapause eggs: female moths treated with this method of removing the subosophageal ganglion (SG removal method) stopped laying eggs. The reason for this was thought to be that SG removal not only suppressed the secretion of diapause hormone, but also inhibited signal transduction that activates egg-laying behavior.

[0017] Therefore, the inventors have conducted extensive research to solve this newly emerged problem, and as a result, they have succeeded in solving the problem of not laying eggs by contacting SG-removed female moths with a solution containing a muscarinic acetylcholine receptor agonist, and producing the desired genetically modified silkworms from any parent lineage. Furthermore, by adding parthenogenetic induction to the above method and producing genetically modified cloned silkworms, they have solved the problem of establishing a genetically stable lineage. The present invention is based on the above research results and provides the following.

[0018] (1) A method for producing a genetically modified silkworm, comprising: a removal step of removing the subesophageal ganglion from a female silkworm; a mating step of mating the female silkworms after the removal step; an egg-laying induction step of contacting or introducing a muscarinic acetylcholine receptor agonist into the female silkworms after the mating step to induce egg-laying; an egg-collection step of collecting fertilized eggs from the female silkworms after the egg-laying induction step; a nucleic acid introduction step of introducing a nucleic acid of interest into the fertilized eggs obtained in the egg-collection step; and a selection step of selecting genetically modified silkworms from the next generation obtained by mating individuals developed from the non-diapause eggs after the nucleic acid introduction step. (2) The method according to (1), wherein the silkworms used in the removal step are diapause strain silkworms. (3) The method for producing an egg-laying egg, as described in (1) or (2), wherein the muscarinic acetylcholine receptor agonist in the egg-laying induction step is selected from the group consisting of pilocarpine, cevimeline, carpronium, xanomeline, carbachol, bethanechol, aceclidine, albamelin maleate, and aclatonium napadisilate. (4) The method according to any one of (1) to (3), further comprising introducing a marker gene in the nucleic acid introduction step. (5) The method according to (4), wherein the selection in the selection step is based on the expression of the marker gene. (6) A method for producing a genetically modified cloned silkworm, comprising: a removal step of removing the subesophageal ganglion from a female individual of a parthenogenetic strain silkworm; an unfertilized egg collection step of harvesting non-dormant unfertilized eggs from the silkworm after the removal step; a parthenogenetic induction step of subjecting the non-dormant unfertilized eggs obtained in the unfertilized egg collection step to a parthenogenetic induction treatment; a nucleic acid introduction step of introducing a nucleic acid of interest into the non-dormant unfertilized eggs after the parthenogenetic induction step; and a selection step of selecting a genetically modified individual as a genetically modified cloned silkworm from individuals developed from the non-dormant eggs after the nucleic acid introduction step. (7) A method for producing a genetically modified cloned silkworm, comprising a removal step of removing the subesophageal ganglion from a female silkworm; a mating step of mating the female silkworms after the removal step; an egg-laying induction step of inducing egg-laying by contacting or introducing a muscarinic acetylcholine receptor agonist into the female silkworms after the mating step; an egg-collection step of collecting fertilized eggs from the female silkworms after the egg-laying induction step; 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 the female silkworms obtained from the fertilized eggs after the nucleic acid introduction step; a parthenogenetic induction step of performing a parthenogenetic induction treatment on the unfertilized eggs obtained in the unfertilized egg collection step; and a selection step of selecting a genetically modified cloned silkworm from the individuals generated after the parthenogenetic induction step. The method of producing said product. (8) The method according to (7), wherein the silkworms used in the removal step are diapausing silkworms. (9) The method for producing an egg-laying egg, as described in (7) or (8), wherein the muscarinic acetylcholine receptor agonist in the egg-laying induction step is selected from the group consisting of pilocarpine, cevimeline, carpronium, xanomeline, carbachol, bethanechol, aceclidine, albamelin maleate, and aclatonium napadisilate. (10) The method according to any one of (6) to (9), wherein the silkworms in the removal step are final-stage larvae or pupae. (11) The method according to any one of (6) to (10), further comprising introducing a marker gene in the nucleic acid introduction step. (12) The method according to (11), wherein the selection in the selection step is based on the expression of the marker gene. (13) The method according to any one of (6) to (12), wherein the parthenogenetic induction treatment is a high-temperature treatment in which an unfertilized egg is exposed to 45°C to 50°C for 15 to 20 minutes. Effect of the Invention

[0019] According to the method for producing genetically modified silkworms of the present invention, it is possible to obtain non-diapause eggs from any diapause parent lineage, and by microinjection into these non-diapause eggs, genetically modified silkworms can be produced with a high hatching rate.

[0020] Furthermore, by making the transgenic silkworms into transgenic cloned silkworms, it is possible to maintain the lineage in a genetically stable state while reducing the effort and cost. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a flow diagram of a method for producing a transgenic silkworm of the present invention. [Diagram 2] FIG. 1 is a flow chart showing a first method for producing a genetically modified cloned silkworm of the present invention. [Diagram 3] FIG. 2 is a flow chart of a second method for producing a genetically modified cloned silkworm of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] 1. Method for producing transgenic silkworms 1-1. Overview The first aspect of the present invention relates to a method for producing a transgenic silkworm. According to the method of the present invention, a transgenic silkworm is produced using a non-diapause egg obtained by contacting a female silkworm moth from which SG has been removed with a solution containing a muscarinic acetylcholine receptor agonist. According to the method of the present invention, it is possible to produce a transgenic silkworm from any parent line suitable for industrial use.

[0023] 1-2.Definition As used herein, the following terms are defined. "Genetic recombination" refers to artificially modifying the natural genetic information of a host organism. Modification of genetic information here includes addition, deletion, and substitution of genetic information. Artificial modification of genetic information can be achieved by existing genetic recombination techniques. For example, a method of adding genetic information not possessed by a host organism using a vector such as a plasmid or a transposon, or a method of destroying genetic information possessed by a host organism, can be mentioned. In addition, in this specification, genome editing techniques that modify the genome information of a host organism by genome editing are also included as genetic recombination in a broad sense.

[0024] "Genetically modified silkworm" (transgenic silkworm) refers to a genetically modified silkworm or its progeny produced using genetic engineering technology. In this specification, genetically modified silkworm refers to, but is not limited to, a genetically modified silkworm obtained by introducing foreign DNA into silkworm eggs by microinjection.

[0025] The term "cloned silkworm" refers to silkworm individuals that have the same genome composition and that constitute a genetically homogeneous population with the same gene composition. In general, a silkworm population obtained by inducing parthenogenesis on unfertilized eggs obtained from a parthenogenetic lineage silkworm, which will be described later, corresponds to a cloned silkworm.

[0026] 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.

[0027] In this specification, a "lineage" is a group of individuals having a specific genetic trait common to the same species, and is almost the same concept as a "strain". In this specification, a lineage also includes a group of mutants having a mutation in a specific gene and a variety having a common morphology or property. In principle, the biological species targeted in this specification is a silkworm. Therefore, unless otherwise specified, the lineage in this specification means a "silkworm lineage", and a silkworm individual belonging to each lineage is called a "lineage silkworm". For example, the dormant lineage described later is a dormant lineage of silkworms, that is, a "dormant silkworm lineage", and a silkworm belonging to this dormant silkworm lineage is called a "dormant lineage silkworm". Note that when focusing on different genetic traits, one individual may belong to multiple lineages. For example, a dormant parthenogenetic lineage has two genetic traits, dormancy and parthenogenesis, so when focusing on dormancy, it belongs to a dormant lineage, and when focusing on parthenogenesis, it belongs to a parthenogenetic lineage.

[0028] As used herein, the term "dormancy" refers to a state in which an organism temporarily ceases development, growth, or activity at a specific time during its life cycle and goes into a dormant state.

[0029] In this specification, "diapause eggs" refers to eggs obtained from diapause strain silkworms and have the potential to transition to a diapause state. It does not matter whether or not the eggs have actually transitioned to a diapause state. Usually, diapause eggs can be obtained by making adult female diapause strain silkworms lay eggs under general rearing conditions. The diapause eggs stop embryo development at the embryo stage about two days after laying and enter a diapause state with low temperature resistance (Yaninuma Toshinobu, 2015, Silkworm and Insect Biotech, 84: 100). This is thought to be one of the life cycle control phenomena based on the environmental response that silkworms have acquired to survive the year. Therefore, many of the derived lineages that have an ancestral lineage in a temperate zone where winter generally exists are diapause lineages.

[0030] In this specification, "diapause (silkworm) lineage" refers to an individual population that lays diapause eggs with respect to a specific genetic trait called egg dormancy. Silkworms are classified into univoltine lineages, bivoltine lineages, and multivoltine lineages. Of these, many univoltine lineages are diapause silkworm lineages that lay diapause eggs. A "univoltine lineage" is a lineage in which adults emerge once a year when reared under natural conditions, a "bivoltine lineage" is a lineage in which adults emerge twice a year, and a "multivoltine lineage" is a lineage in which adults emerge multiple times a year. In general, many of the derived lineages that have an ancestral lineage in a temperate region where winter exists are univoltine lineages. However, even diapause silkworm lineages may be able to lay non-diapause eggs by the non-diapause egg-laying treatment described below.

[0031] As used herein, the term "non-diapause eggs" refers to silkworm eggs that do not have the potential to become diapause. Examples of such eggs include non-diapause eggs obtained from a multivoltine strain, non-diapause eggs obtained from a diapause silkworm strain by a non-diapause egg-laying treatment, and non-diapause eggs obtained by the method described in Japanese Patent No. 6765803.

[0032] As used herein, "non-diapause (silkworm) strain" refers to an individual population that lays non-diapause eggs that do not enter a diapause state during the egg-laying stage. In general, 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 Ringetsu.

[0033] As used herein, "parthenogenetic (silkworm) line" refers to a line in which parthenogenesis is induced with high efficiency. In parthenogenetic silkworm line, parthenogenesis is induced by applying physical or chemical stimuli to unfertilized eggs. In general, most silkworm lineages capable of parthenogenesis are dormant silkworm lineages. Therefore, in this specification, parthenogenetic silkworm lineages are also referred to as "dormant parthenogenetic (silkworm) lineages."

[0034] "Diapause hormone" is a hormone consisting of a neuropeptide consisting of 24 amino acids with an amide-type C-terminus. Diapause hormone is synthesized in diapause factor (DF) cells in the subesophageal ganglion during the pupal stage of the silkworm mother moth. Diapause hormone is transported to the corpus allata (corpus cardiaca) via the periesophageal nerve cord, etc. It is then released from the corpus allata into the body fluids during the early pupal stage and acts on the ovaries, causing the eggs laid by the mother moth to become diapause eggs.

[0035] The "suboesophageal ganglion" (often abbreviated as "SG" in this specification) is an organ that synthesizes diapause hormone during the pupal stage of the mother moth in response to the environment experienced during the egg and larval stages, and transports it to the corpus allata, a secretory organ. If this suboesophageal ganglion is removed, diapause hormone will not be synthesized, and all eggs will become non-diapause eggs.

[0036] 1-3. Preparation method The flow of the method for producing a genetically modified silkworm according to this embodiment is shown in Figure 1. As shown in Figure 1, this production method includes a removal step (S0101), a mating step (S0102), an egg-laying induction step (S0103), an egg collection step (S0104), a nucleic acid introduction step (S0105), and a selection step (S0106) as essential steps. Each step will be described in detail below.

[0037] (1) Removal process The "removal step" (S0101) is a step of removing the subesophageal ganglion (SG) from female silkworms. The present invention aims to produce transgenic silkworms from any parent line. Therefore, the silkworm line used in this step is not limited. However, considering that one of the objects of the present invention is to obtain non-diapause eggs required for carrying out the microinjection method from a diapause line suitable for industrial use, a diapause line of silkworms is preferred.

[0038] The dormant silkworm strain used in this process is not particularly limited. Examples of dormant silkworm strains include Daizo, J137, Shi146, J603, J604, Chu604, Chu605, Chu514, Chu515, Chu9.0, J9.0, Shunrei, Kanetsuki, Hitachi, Nishiki, J502, Shi146, Shi122, Tokushi2, Ou7, Tokushi4, and Kakushina, but any of them may be used.

[0039] In this process, 4th instar larvae, final instar larvae (including pre-pupal stage), and pupal stage silkworm female individuals are used. Although not limited to these, the use of final instar larvae and pupae is preferred. In particular, the use of early pupae, specifically pupae within 48 hours, 36 hours, or 24 hours after pupation, is preferred. This is because eggs laid by female individuals treated with SG removal at this stage are almost 100% non-diapause eggs.

[0040] The removal operation is not particularly limited as long as the silkworm can continue to develop after this process. Usually, a method of extracting the SG by a surgical procedure is used. The method of extracting the SG is not particularly limited, but for example, after anesthetizing the individual to be removed (e.g., a pupa), a small incision is made at the tip of the pupa's head with a scalpel, and the incision is opened with forceps. The purple SG is pinched with tweezers and pulled out from the incision, and the nerve axons connecting the SG to the brain and abdominal ganglion are cut to extract only the SG. The anesthesia method is not limited. For example, water anesthesia by immersion in water or ether anesthesia may be used. After the SG is extracted, the body fluids overflowing from the incision are wiped off with a cotton swab, and the wound is closed with dissolved paraffin. After this step, the individuals can be left at rest at 25°C until they emerge.

[0041] (2)Mating process The "mating step" (S0102) is a step of mating the female adult insects after the removal step with male adult insects. The lineage of the male insects used for mating is not particularly limited. Silkworm mating can be carried out according to standard methods. In general, females are able to mate immediately after emergence. Mating time can be 2 to 3 hours. Usually, mating once is sufficient. It is also possible to place healthy female moths that have not undergone the SG process to promote the mating behavior of male moths.

[0042] (3) Spawning induction process The "spawning induction step" (S0103) is a step of inducing female individuals after the mating step (S0102) to spawn. Eggs obtained from female moths from which the SG has been removed in the removal step are all non-diapause eggs because the female moths do not synthesize diapause hormone. However, the inventors' experiments have revealed that female moths from which the SG has been removed have inactivated egg-laying behavior and no longer lay eggs. This has resulted in an essential problem in that the desired non-diapause fertilized eggs cannot be obtained. Therefore, this step is a step in which a treatment is carried out to stimulate egg-laying in female moths that have undergone the removal step and mating step, and to activate the egg-laying behavior. Therefore, this step, along with the removal step (S0101), is one of the most important components of the method for producing genetically modified silkworms of this embodiment.

[0043] As used herein, "oviposition induction" refers to artificially treating female moths whose oviposition behavior has been suppressed or inhibited by SG removal to induce oviposition behavior and promote oviposition.

[0044] The method for inducing egg-laying induction may involve contacting or introducing a muscarinic acetylcholine receptor agonist into female individuals after the mating step.

[0045] "Muscarinic acetylcholine receptor (mAChR)" refers to a G protein-coupled acetylcholine receptor (AChR) present on the plasma membrane of certain neurons and other cells. It is the primary endoreceptor stimulated by acetylcholine released from postganglionic fibers of the parasympathetic nervous system.

[0046] "Muscarinic acetylcholine receptor agonist (mAChR agonist)" refers to a substance that binds to mAChR and acts as an agonist (agonist). Specific examples include acetylcholine and muscarine, as well as non-selective mAChR agonists such as pilocarpine, cevimeline, carpronium, xanomeline, carbachol, bethanechol, aceclidine, albamelin maleate, and aclatonium napadisilate.

[0047] The method of contacting the mAChR agonist with the female moth is not limited as long as the mAChR agonist contacted with the surface of the silkworm body binds to the mAChR of the silkworm and activates it to induce egg-laying. For example, the method includes immersing all or a part of the female moth in a solution in which the mAChR agonist is dissolved or suspended in a solvent, or applying or spraying the solution on all or a part of the female moth. The "part" referred to here is not limited as long as it is a part of the surface of the female moth's body, but is preferably the abdomen, and more preferably the tail including the egg-laying opening at the tip of the abdomen.

[0048] The method for introducing the mAChR agonist into the female moth is not limited, but may be, for example, a solution in which the mAChR agonist is dissolved or suspended in a solvent, which may be introduced into the female moth by, for example, microinjection.

[0049] When a solution containing an mAChR agonist is used, the concentration of the mAChR agonist is not limited, but may be 0.001% or more, 0.01% or more, 0.1% or more, or 1% or more. The contact time is not limited, and may be, for example, 10 seconds or less, 50 seconds or less, 30 seconds or less, 40 seconds or less, 50 seconds or less, or 60 seconds or less.

[0050] (4) Egg collection process The "egg collection step" (S0104) is a step of collecting fertilized eggs from female individuals after the egg-laying induction step (S0103). The method of collecting eggs in this step is not limited. For example, a method of allowing a female moth to naturally lay eggs according to a known egg collection method can be used. Specifically, for example, a method is employed in which an egg-laying mat is provided to a female moth after mating, and the female moth is allowed to lay eggs on the mat, and eggs are collected. Usually, egg-laying often begins several hours after the mating mat is provided. As mentioned above, the female moth used in this method will not lay eggs unless it has gone through the egg-laying induction step (S0103). Therefore, the female moth is provided with an egg-laying mat after the egg-laying induction step.

[0051] In order to efficiently incorporate the nucleic acid of interest introduced into the fertilized eggs in the nucleic acid introduction step (S0105) described below into the nucleus, it is desirable to start the nucleic acid introduction within 2 to 8 hours, preferably 3 to 6 hours, after laying, so in this step, it is preferable to have the female individuals lay eggs in accordance with the timing of the nucleic acid introduction. This can be achieved by placing the female moth after the egg-laying induction step (S0103) at a low temperature of 0 to 10°C, preferably 5°C, storing it at that temperature for 1 to 2 days, and up to 1 to 7 days, and then transferring the female moth from the low temperature to room temperature (23 to 28°C), providing an egg-laying mat there, and allowing it to start laying eggs under dark conditions. Since silkworm embryo development begins immediately after laying, this method makes it possible to stably obtain fertilized eggs in this step at a stage of embryo development suitable for nucleic acid introduction.

[0052] In addition, the eggs collected in this step are non-diapause fertilized eggs in principle, since the SGs are removed in the removal step (S0101) and then mated female moths are used in the mating step (S0102).

[0053] (5) Nucleic acid introduction step The "nucleic acid introduction step" (S0105) is a step of introducing a nucleic acid of interest into the non-diapausing unfertilized eggs after the egg collection step (S0104). In this step, the "nucleic acid of interest" refers to a nucleic acid to be introduced into silkworms to produce the desired genetically modified 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 the gene of interest, an expression vector containing the gene, and a helper plasmid. When a gene is introduced, 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 imparting a desired trait can be introduced. At this time, a marker gene may be introduced so that an individual into which the nucleic acid of interest has been introduced can be easily selected in the selection step described below.

[0054] As used herein, the term "marker gene" refers to a polynucleotide consisting of a base sequence that codes for a marker protein, also called a selection marker.

[0055] As used herein, the term "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. A labeled protein that is less invasive to the transformant upon detection is preferred. Examples of such a labeled protein include fluorescent proteins, pigment synthesis proteins, luminescent proteins, exocytic proteins, and proteins that control the external morphology. Fluorescent proteins, pigment synthesis proteins, luminescent proteins, and exocytic 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 are easy to distinguish and select the transformant.

[0056] 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. In addition, there are no particular limitations on the excitation wavelength or fluorescence wavelength. Specific examples include CFP, RFP, DsRed, YFP, PE, PerCP, APC, GFP, etc.

[0057] As used herein, a "pigment synthesis protein" is a protein involved in the biosynthesis of a pigment, and is usually an enzyme. The "pigment" referred to here is a low molecular weight compound or peptide capable of imparting a pigment to a transformant, regardless of the type. Preferably, the pigment is one that appears as the external color of an individual. Examples include melanin pigments (including dopamine melanin), ommochrome pigments, and pteridine pigments.

[0058] As used herein, the term "luminescent protein" 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.

[0059] As used herein, the term "exocrine protein" refers to a protein secreted outside a cell or outside the body, and includes exocrine enzymes, 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.

[0060] The origin of the target nucleic acid introduced in this step is not limited to any particular organism. It may be derived from the host silkworm, or may be a gene derived from another organism, such as a human. For example, when producing an antibody using a genetically modified silkworm, the foreign DNA may be an expression unit including an IgG antibody gene and a gene expression regulatory region such as a promoter or terminator.

[0061] In this step, a peptide (including a protein such as an enzyme) and / or a low molecular weight compound can be introduced together with the nucleic acid, if necessary. In this case, the order of introduction with respect to the nucleic acid does not matter. It may be before introduction of the nucleic acid, after introduction of the nucleic acid, or simultaneously with introduction of the nucleic acid.

[0062] This step can be carried out by a method known in the art for introducing a foreign gene into a silkworm. For example, if the expression vector is a plasmid having inverted terminal repeats of a transposon 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 a buffer to an appropriate concentration to prepare an injection solution. At this time, a helper plasmid having a DNA encoding a transposon transferase is added to the injection solution. An example of the helper plasmid is pHA3PIG.

[0063] The introduction of the target nucleic acid into the egg is generally performed using a special injection device that utilizes air pressure, although there is no limitation thereto. For example, the method of 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 the nucleic acid to be introduced is not particularly limited. It may be appropriately determined depending on the type, properties, and purpose of the nucleic acid. It is usually 1 nL to 5 nL.

[0064] After the introduction of the nucleic acid, the eggs may be incubated under appropriate conditions, for example, at 25° C., until they hatch. All individuals that hatch after this step are cloned silkworms capable of parthenogenesis.

[0065] (6) Selection process The "selection step" (S0106) is a step of selecting genetically modified organisms from the next generation (G1) obtained by mating individuals that have developed from the non-diapause eggs after the nucleic acid introduction step (S0105). The silkworms obtained after this step are the desired genetically modified silkworms.

[0066] 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 (S0105). For example, when the nucleic acid of interest is a foreign gene, genomic DNA or mRNA may be prepared from silkworms generated after the nucleic acid introduction step (S0105), and the presence or absence of the foreign gene or the expression of the foreign gene may be confirmed by PCR or the like.

[0067] Alternatively, when the expression vector used for nucleic acid introduction contains a marker gene, the desired genetically modified silkworm can be selected by expression of the marker gene. The marker protein produced by expression of the marker gene can impart a new trait that the host silkworm does not have. Based on the activity of this marker protein, it becomes possible to easily distinguish a transformant that has the introduced nucleic acid of interest. Here, "based on activity" means based on the result of activity detection. The activity may be detected by directly detecting the activity of the marker protein itself, or indirectly detecting the activity via a metabolite generated by the activity of the marker protein, such as a dye. The detection may be chemical detection (including enzyme reaction detection), physical detection (including behavioral analysis detection), or sensory detection by the person detecting it (including detection by vision, touch, smell, hearing, and taste).

[0068] 2. Method for producing genetically modified cloned silkworms 2-1. Overview A second aspect of the present invention relates to a method for producing a transgenic cloned silkworm.

[0069] The production methods of the present invention include a first production method in which female parthenogenetic silkworms are subjected to an SG removal treatment and non-diapause eggs obtained from ovarian eggs are used to produce genetically modified cloned silkworms, and a second production method in which genetically modified cloned silkworms are produced using non-diapause eggs obtained by contacting SG-removed female moths with a solution containing a muscarinic acetylcholine receptor agonist.

[0070] When genetically modified silkworms produced by the genetically modified silkworm production method described in the first aspect are repeatedly crossed over a long period of time to maintain the lineage, it may become difficult to ensure genetic stability within the same lineage. However, with the genetically modified cloned silkworm of this aspect, it is possible to produce genetically modified cloned silkworms using non-diapause eggs obtained from female moths that have been subjected to the SG removal treatment of the first aspect, and to maintain the lineage in a genetically stable state. This solves the above-mentioned problems in the first aspect of the invention, and provides a genetically modified silkworm that can produce stable quality and protein regardless of the lineage or individual differences of the transformant.

[0071] 2-2. Manufacturing method 2-2-1. First manufacturing method The flow of the first method for producing a genetically modified cloned silkworm is shown in FIG. 2. As shown in FIG. 2, the first production method includes a removal step (S0101), an unfertilized egg collection step (S0107), a parthenogenetic induction step (S0108), a nucleic acid introduction step (S0105), and a selection step (S0106) as essential steps. Among these, the removal step (S0101), the nucleic acid introduction step (S0105), and the selection step (S0106) are the same as the respective steps described in the above-mentioned first embodiment. However, some steps are unique to the production method of the second embodiment. The unfertilized egg collection step (S0107) and the parthenogenetic induction step (S0108) are steps unique to the production method of a genetically modified cloned silkworm.

[0072] Below, the unfertilized egg collection step (S0107) and the parthenogenesis induction step (S0108) will be specifically explained, and for the steps that are the same as those in the first embodiment, only the points unique to the production method of the second embodiment will be explained.

[0073] (1)Removal process The "removal step" (S0101) is a step of removing the subesophageal ganglion (SG) from female silkworms. The silkworm strain used in this step is a parthenogenetic silkworm strain. Any parent strain can be used in the removal step in the method for producing a transgenic silkworm of the first embodiment, but the removal step of this embodiment is different in this respect.

[0074] In addition, either a diapausing strain of silkworms or a non-diapausing strain of silkworms may be used, but a diapausing strain of silkworms is preferred for the same reasons as in the method for producing a genetically modified silkworm of the first embodiment.

[0075] (2) Unfertilized egg collection process The "unfertilized egg collection step" (S0107) is a step of collecting non-diapausing unfertilized eggs from the parthenogenetic silkworm strain silkworms after the removal step (S0101). In the first embodiment of the method for producing a genetically modified silkworm, when obtaining non-diapause eggs from a female moth whose egg-laying behavior has been inactivated by SG removal treatment, an egg-laying induction step (S0103) is carried out to induce the female moth to lay eggs, and the female moth is allowed to lay eggs. However, in the first production method of this embodiment, since the female moth is a parthenogenetic strain silkworm, non-diapause eggs can be obtained by directly collecting ovarian eggs from a female moth that has been subjected to SG removal treatment, without going through the mating step (S0102), egg-laying induction step (S0103), and egg collection step (S0104). In this respect, the method is significantly different from the first embodiment of the method for producing a genetically modified silkworm.

[0076] In this step, individuals in which unfertilized eggs are capable of parthenogenesis in the body, i.e., individuals that have developed into mature unfertilized eggs, are used. Adult individuals that have emerged after the removal step may be used. Preferably, the adults are refrigerated at a low temperature of 0 to 10°C, preferably 5°C, immediately after emergence or within 2 days after emergence, and the refrigeration period is within 10 days.

[0077] After the removal process, the females do not lay eggs due to the inactivation of egg-laying behavior. Therefore, the method of collecting unfertilized eggs is, in principle, a method of collecting them by dissection. Specifically, for example, after incising the abdomen or tail of the female adult with a scalpel or dissecting scissors, pressure is applied to the abdomen with a finger to remove the ovaries from the incision. The aggregated oviduct can be loosened by immersing the removed ovaries in water. Next, the ovaries are placed on a fine stainless steel mesh or gauze, and rubbed with a finger or the like to separate the oviduct tissue and the unfertilized eggs. Finally, the oviduct tissue floating on the water is removed by pouring water through the mesh, and the unfertilized eggs remaining at the bottom are collected. This operation can be repeated several times to collect only the unfertilized eggs.

[0078] (3) Parthenogenesis induction process The "parthenogenesis induction step" (S0108) is a step of inducing parthenogenesis in the non-dormant unfertilized eggs obtained in the unfertilized egg collection step (S0107). This step is carried out in combination with the unfertilized egg collection step, and the obtained silkworms are cloned silkworms.

[0079] The parthenogenetic induction treatment may be performed 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 Experimental Research Report, 24: 1-6), and Kosegawa (E. Kosegawa et al., 2012, J Insect Biotechnol Sericology, 81: 37-44) may be referred to. In general, parthenogenetic induction is induced by applying physical or chemical stimuli to unfertilized eggs. Specifically, for example, high temperature treatment may be mentioned. As a specific example of high temperature treatment, the unfertilized eggs collected in the above step may be exposed to hot water treatment at 45°C to 50°C for 15 to 20 minutes. After that, it is preferable to keep them at 12°C to 18°C ​​for 2 to 6 days.

[0080] (4) Nucleic acid introduction step The "nucleic acid introduction step" (S0105) is a step of introducing a target nucleic acid into a non-dormant unfertilized egg after the parthenogenesis induction step (S0108). The eggs used for nucleic acid introduction in this step are non-dormant fertilized eggs obtained in the unfertilized egg collection step (S0107), and are different from the nucleic acid introduction step (S0105) of the first embodiment in that they are not fertilized eggs that have been through the mating step (S0102). However, other basic procedures are the same as the nucleic acid introduction step (S0105) described in the first embodiment, and therefore a detailed description here will be omitted.

[0081] (5) Selection process The "selection step" (S0106) is a step of selecting genetically modified organisms from individuals that have developed from non-diapause eggs after the nucleic acid introduction step (S0105). Although parthenogenetic strains are themselves clones, the individuals that hatch after the nucleic acid introduction step have their genomes altered, and each individual becomes a separate first generation clone. The next generation, which are collected from the first generation, parthenogenetically develop eggs, and are obtained through the selection of genetically modified organisms, are all cloned silkworms, as described above. Therefore, the silkworms obtained after this step are the desired genetically modified cloned silkworms.

[0082] The basic procedure of this step is the same as the selection step (S0106) described in the first embodiment, so a detailed description thereof will be omitted here.

[0083] 2-2-2. Second manufacturing method The flow of the second method for producing a genetically modified cloned silkworm is shown in Figure 3. As shown in Figure 3, the second production method includes a removal step (S0101), a mating step (S0102), an egg-laying induction step (S0103), an egg collection step (S0104), a nucleic acid introduction step (S0105), an unfertilized egg collection step (S0107), a parthenogenetic induction step (S0108), and a selection step (S0106) as essential steps. Among these, the removal step (S0101), the mating step (S0102), an egg-laying induction step (S0103), an egg collection step (S0104), a nucleic acid introduction step (S0105), and a selection step (S0106) are basically the same as the respective steps of the first embodiment. In addition, the unfertilized egg collection step (S0107) and the parthenogenetic induction step (S0108) are basically the same as the respective steps of the first production method. Hereinafter, for each step, the parts that overlap with the first embodiment or the first production method will be omitted, and only the parts that are unique to the second production method will be explained.

[0084] (1)Removal process The basic procedure of the "removal step" (S0101) is the same as the nucleic acid introduction step (S0105) described in the first embodiment. Moreover, the silkworms used in this step are different from the removal step of the first production method in that they are not, in principle, typical parthenogenetic strain silkworms that are maintained as clones by parthenogenesis. Furthermore, the silkworms used in this step may be either dormant strain silkworms or non-dormant strain silkworms, but dormant strain silkworms are preferred for the same reasons as in the method for producing genetically modified silkworms of the first embodiment.

[0085] (2)Mating process The "mating step" (S0102) is a step of mating the female adult insects after the removal step with male adult insects. The basic procedure of this step is the same as the mating step (S0102) described in the first embodiment, so a detailed description thereof will be omitted here.

[0086] (3) Spawning induction process The "spawning induction step" (S0103) is a step of inducing female individuals after the mating step (S0102) to spawn. The basic procedure of this step is the same as the spawning induction step (S0103) described in the first embodiment, so a detailed description thereof will be omitted here.

[0087] (4) Egg collection process The "egg collection step" (S0104) is a step of collecting fertilized eggs from female individuals after the egg-laying induction step (S0103). The basic procedure of this step is the same as the egg collection step (S0104) described in the first embodiment, so a detailed description thereof will be omitted here.

[0088] (5) Nucleic acid introduction step The "nucleic acid introduction step" (S0105) is a step of introducing a target nucleic acid into the fertilized eggs obtained in the egg collection step. The basic method of this step is the same as the nucleic acid introduction step (S0105) described in the first embodiment, so a detailed description will be omitted here. After the nucleic acid introduction, the silkworm eggs are incubated under appropriate conditions, for example, at 25° C. until they hatch, and then reared according to a conventional method until they become adults.

[0089] (6) Unfertilized egg collection process The unfertilized egg collection step (S0107) is a step of collecting unfertilized eggs from the female individuals obtained from the fertilized eggs after the nucleic acid introduction step (S0105).

[0090] The basic method of this step is similar to the method described in the first production method, but differs from the unfertilized egg collection step of the first production method in that, whereas in the first production method, unfertilized eggs are collected from G0 silkworms of a parthenogenetic silkworm strain that has been subjected to the removal step (S0101), in this step of the second production method, unfertilized eggs are collected from G1 female individuals obtained from fertilized eggs after the nucleic acid introduction step.

[0091] As described above, the female individuals used are individuals in which unfertilized eggs have developed to a state in which parthenogenesis is possible within the body.

[0092] The method for collecting unfertilized eggs from female individuals is not limited, and examples thereof include a method for collecting eggs by dissection or a method for collecting eggs by natural spawning.

[0093] The method for collecting the eggs by dissection is similar to the unfertilized egg collection step described in the first production method.

[0094] The method of collecting eggs by natural egg collection can be achieved by making unmated female silkworms lay unfertilized eggs after emergence. The specific method of making unmated female silkworms lay unfertilized eggs is similar to the egg collection step described in the first production method.

[0095] (7) Parthenogenesis induction process The parthenogenesis induction step (S0108) is a step of performing parthenogenesis induction treatment on the unfertilized eggs obtained in the unfertilized egg collection step (S0107). The method for this step may be similar to that described in the first preparation method.

[0096] (8) Selection process The selection step (S0106) is a step of selecting genetically modified individuals as genetically modified cloned silkworms from the individuals generated after the parthenogenesis induction step. The method of this step may be similar to the method described in the first embodiment. EXAMPLES

[0097] The present invention will be specifically described below with reference to examples. However, the following examples are merely illustrative of one embodiment of the present invention and are not intended to limit the scope of the present invention.

[0098] <Example 1> (the purpose) We will verify whether muscarinic acetylcholine receptor agonist treatment increases egg production in SG-deprived female moths.

[0099] Materials and Methods (1) Silkworm strains and their rearing The silkworm strain used was the bivoltine non-diapause strain "Ariake," obtained from the National Agriculture and Food Research Organization (NARO). The eggs were incubated in the dark at 25°C. The hatched silkworms were raised on artificial food (Nihon Nosan Kogyo) at 27-28°C until the third instar, and from the fourth instar onwards, they were raised at 25°C until they reached adulthood. The daily light / dark period from hatching to the adult stage was 16 hours light and 8 hours dark.

[0100] (2)SG removal SG removal was performed on pupae within 1 day of pupation. The pupae used were obtained from cocoons 5 days after the start of spinning of 1-week-old larvae. To remove SGs, a small incision was made at the tip of the pupal head with a scalpel, the incision was opened with forceps, and the purple SGs were pulled out from the incision under a stereomicroscope and excised entirely with tweezers. After removing the SGs, the incision was covered with molten paraffin and incubated at 25°C until emergence. The emerged female moths were used for the next step.

[0101] (3) Mating and spawning induction treatment The SG-removed female moths and intact male moths were mated for 3-4 hours at 25°C, and then incubated overnight at 5°C while mated. The male moths were then removed, and some of the female moths were placed on egg-laying mats and kept in a dark room at 25°C. The number of eggs laid by the next day was counted. Oviposition induction treatment was performed by immersing the tail of the female moth in a solution of pilocarpine hydrochloride (FUJIFILM Wako Chemical Co., Ltd.) adjusted to 0.5% with sterilized distilled water. Thereafter, the female moths were placed on an egg-laying mount and allowed to lay eggs in a dark room at 25° C. The number of eggs laid by the next day was counted.

[0102] (result) The results are shown in Table 1.

[0103] [Table 1]

[0104] The average number of eggs laid in Experiment No. 2, which consisted of a group of SG-removed females treated with pilocarpine hydrochloride, a muscarinic acetylcholine receptor agonist, was more than 10 times that laid in Experiment No. 1, which consisted of a group of untreated SG-removed females. This result demonstrated that even SG-removed female moths are able to lay eggs when exposed to a solution containing a muscarinic acetylcholine receptor agonist.

[0105] <Example 2> (the purpose) A transgenic silkworm is produced using the production method according to the first aspect of the present invention.

[0106] Materials and Methods (1) Silkworm strains, their rearing, and SG removal These methods are the same as those in the first embodiment, so the explanation will be omitted here.

[0107] (2) Mating and spawning induction treatment The SG-removed female moths and intact male moths were mated for 3-4 hours at 25°C, and then incubated overnight at 5°C while mated. The male moths were then removed, and some of the female moths were placed on egg-laying mats and kept in a dark room at 25°C. The number of eggs laid by the next day was counted. For the egg-laying induction treatment, a solution of pilocarpine hydrochloride (FUJIFILM Wako Chemical Co., Ltd.) adjusted to 0.01% and 0.1% with sterilized distilled water was used. Male and female moths were separated and transferred from 5°C to 25°C, and 20 μL of the pilocarpine hydrochloride solution was injected into the abdomen of the female moth. Thereafter, the female moths were placed on an egg-laying mount and allowed to lay eggs in a dark room at 25° C. The number of eggs laid by the next day was counted.

[0108] (3) Nucleic acid introduction The eggs used for nucleic acid introduction were F1 fertilized eggs that had been laid on egg-laying mats in a dark room at 25°C after post-spawning treatment and were 0 to 2 hours after spawning. The F1 fertilized eggs were attached to a slide glass with adhesive, and the target gene, pBac3xP3DsRed (160ng / μL), was mixed with transposon transferase mRNA (80ng / μL) and the same enzyme Helper DNA (160ng / μL), and introduced by microinjection. The gene recombination solution for microinjection was prepared using the HiSpeed ​​Plasmid Midi Kit (Qiagen).

[0109] For genome editing, a mixture of crRNA (od-2; Daimon T., et al., 2013, Dev. Growth Differ., 56(1): 14-25) and tracrRNA designed to target the gene (BmBLOS2) responsible for the epidermis-transparent mutation (oil silkworm) was heated at 95°C for 5 minutes, then left at room temperature for 15 minutes, and then mixed with Cas9 enzyme and left at room temperature for 15 minutes to prepare an injection solution (final concentration of crRNA, tracrRNA, and Cas9: 200ng / μL each). After microinjection, the eggs were incubated on the slide glass in a humidified state at 25°C until they hatched. The number of hatched larvae was then counted, and the hatching rate was calculated from the number of hatched eggs relative to the number of eggs microinjected (number of eggs introduced).

[0110] (4) Selection of transformants Transgenic silkworms transformed by gene transfer were selected. Hatched larvae were transferred to a container, which was a silkworm bed, using a feather broom. Plastic lunch boxes (DX-HS8: 222 x 143 x 35 mm; or DX-HS20: 256 x 189 x 35 mm; both Chuo Kagaku) ​​were used to rear 1st to 3rd instar juvenile silkworm larvae. Silkmate Original Species 1-3 S (Nihon Nosan Kogyo) was fed to the larvae arranged on dry-proof paper. In principle, the food was replaced once for the 1st and 2nd instars and once to three times for the 3rd instar, but was replaced as appropriate when food was insufficient or dried out. Old food was removed if there was a large amount of leftover food, but if there was only a small amount leftover, it was left as it was and new artificial feed was added.

[0111] For rearing 4th to 5th instar mature silkworm larvae, containers were used, in which silkworm paper (craft paper), dry-proof paper (paraffin paper), or both were laid in layers inside a nursery box for gardening type 2 (model number 201206; Sanko Co., Ltd.). Depending on the humidity and conditions inside the container, dry-proof paper, acrylic, or mesh lids were used. The number of silkworms in each container was counted from the second day of the 4th instar, and the number of silkworms per container was set at approximately 80 or less according to the size of the container. As with the juvenile silkworm larvae, Silkmate original species 1-3 instar M (Nihon Nosan Kogyo) was cut into strips or cut into pieces and given to the larvae. The feed was changed once a day, as a rule, although it varied depending on the amount of leftover food and the dryness of the food. If there was only a small amount of leftover food, new artificial feed was added by cutting it without removing the old food. The rearing temperature was 28°C for the 1st to 4th instars and 25°C for the 5th instar. Mature 5th instar silkworms were transferred to a cage (transferring larvae to a cage) and allowed to spin cocoons at 25℃. The cocoons were then cut open to remove the pupae, which were then placed in a moth box (a cardboard box for storing pupae and moths) to protect them until they emerged. The transgenic silkworms showed a phenotype in which the ocelli of the eggs and larvae exhibited red fluorescence due to the expression of the introduced 3xP3DsRed, and the transgenic silkworms were selected based on this phenotype.

[0112] (result) The results are shown in Table 2.

[0113] [Table 2]

[0114] From Table 2, it was demonstrated that transgenic individuals can be efficiently produced by introducing the target gene into eggs obtained from the SG-removed Ariake strain. It was also clarified that transgenic silkworms (including genome-edited silkworms) can be efficiently obtained regardless of the nucleic acid introduction method, microinjection method or genome editing method.

[0115] <Example 3> (the purpose) The method according to the second aspect of the present invention is used to produce genetically modified clonal silkworms.

[0116] Materials and Methods (1) Silkworm strains and their rearing The silkworm strain used was the univoltine parthenogenetic diapause strain pK1, which was obtained from the National Agriculture and Food Research Organization (NARO). The eggs were incubated in the dark at 25°C. The hatched silkworms were raised on artificial food (Nihon Nosan Kogyo) at 27-28°C until the third instar, and from the fourth instar onwards, they were raised at 25°C until they reached adulthood. The daily light / dark period from hatching to the adult stage was 16 hours light and 8 hours dark.

[0117] (2) SG removal SG removal was performed according to the method described in “(2) SG removal” in Example 1.

[0118] (3) Preparation of unfertilized eggs After the SG was removed, the moths were raised to adulthood, and unfertilized eggs were obtained from the female moths. Unfertilized eggs were collected by cutting the abdomen of the female moth after emergence with a scalpel, dissecting scissors, or surgical scalpel, and then applying pressure to the incision with a finger or the like to push out the ovaries. The excised ovaries were immersed in water, and the oviducts contained within the ovaries were untied. Next, the obtained ovaries were placed on a stainless steel mesh with a mesh size of 0.5 mm or less, and drained. The ovaries on the stainless steel mesh were rubbed with a finger to separate the oviduct tissue and the unfertilized eggs. Next, water was poured over the stainless steel mesh to drain it. The oviduct tissue floating on the water was removed, and the unfertilized eggs that sank to the bottom of the container were collected. This operation was repeated several times to prepare only unfertilized eggs.

[0119] (4) Induction of parthenogenesis Unfertilized eggs collected from each experimental group were kept at 25°C for 5-8 hours, and then heat-shocked at 46°C for 18 minutes to induce parthenogenesis. After induction, the eggs were immersed in cold water at 15°C for 30 minutes to cool, then air-dried and incubated in an air incubator at 15°C for 16-18 hours. The following nucleic acid transfer was then performed.

[0120] (5) Nucleic acid introduction Nucleic acid introduction into non-diapause unfertilized eggs was performed by microinjection into unfertilized eggs that had been incubated at 15° C. for 16 to 18 hours after the parthenogenesis induction by the heat shock treatment. The microinjection procedure was in accordance with the method described in “(3) Nucleic acid introduction” in Example 2.

[0121] (6) Selection of transgenic cloned silkworms From the parthenogenetic clones of generation 0 (G0) produced from unfertilized eggs, clones transformed by gene transfer (TG clones: transgenic clones) were selected. The selection of the transformant was performed according to the method described in "(5) Selection of the transformant" in Example 1. The transformant obtained after this step is the desired genetically modified cloned silkworm.

[0122] (result) The results are shown in Table 3.

[0123] [Table 3]

[0124] The results in Table 3 show that genetically modified cloned silkworms (or genome-edited silkworms) can be obtained from parthenogenetic diapause strains by SG removal.

[0125] <Example 4> (the purpose) When the target nucleic acid introduced into an egg in the nucleic acid introduction step of the present invention is a genome editing-inducing gene, it is verified that the desired genetically modified cloned silkworm is produced.

[0126] (method) SG was removed from female pupal stage individuals of the dormant parthenogenetic line (pK1). Ovaries were removed from emerged individuals to prepare unfertilized eggs. After parthenogenesis treatment of the unfertilized eggs, TALEN was created to knock out the second white egg (BmW2) gene. TALEN was prepared as mRNA.

[0127] The genome editing procedure involved subjecting pK1 unfertilized eggs to temperature treatment (46°C x 18 minutes, 15°C x 15 minutes), and introducing the above-mentioned TALEN into the eggs two hours after treatment using the microinjection method.

[0128] (result) In the experimental group TMY10 where genome editing was performed using TALEN, more than 30% of the injected unfertilized eggs hatched, and unfertilized eggs were subsequently collected from the emerged female moths. Furthermore, BmW2 gene knockout individuals were confirmed from these unfertilized eggs. These knockout individuals could be maintained by parthenogenesis without sexual reproduction, demonstrating that transgenic cloned silkworms can be produced by subesophageal ganglion ablation treatment even when starting from a diapausing parthenogenetic lineage.

Claims

1. A method for producing a transgenic silkworm, comprising the steps of: a step of removing the subesophageal ganglion from the female silkworm; a mating step of mating the female individuals after the removal step; a spawning induction step of contacting or introducing a muscarinic acetylcholine receptor agonist into the female individual after the mating step to induce spawning; an egg collection step of collecting fertilized eggs from the female individuals after the egg-laying induction step; a nucleic acid introduction step of introducing a nucleic acid of interest into the fertilized eggs obtained in the egg collection step; A selection step of selecting genetically modified silkworms from the next generation obtained by mating individuals developed from the non-diapause eggs after the nucleic acid introduction step. The method of producing said product.

2. The method according to claim 1 , wherein the silkworms used in the removal step are diapausing silkworms.

3. The method for producing an egg-laying egg, according to claim 1 or 2, wherein the muscarinic acetylcholine receptor agonist in the egg-laying induction step is selected from the group consisting of pilocarpine, cevimeline, carpronium, xanomeline, carbachol, bethanechol, aceclidine, albamelin maleate, and aclatonium napadisilate.

4. The method according to claim 1 or 2, further comprising introducing a marker gene in the nucleic acid introduction step.

5. The method according to claim 4 , wherein the selection in the selection step is based on the expression of the marker gene.

6. A method for producing a genetically modified cloned silkworm, comprising the steps of: A step of removing the subesophageal ganglion from a female parthenogenetic silkworm; an unfertilized egg collection step of collecting non-dormant unfertilized eggs from the silkworms after the removal step; a parthenogenetic induction step of subjecting the non-dormant unfertilized eggs obtained in the unfertilized egg collection step to parthenogenetic induction treatment; A nucleic acid introduction step of introducing a nucleic acid of interest into the non-dormant unfertilized egg after the parthenogenetic induction step; and A selection step of selecting genetically modified organisms as genetically modified cloned silkworms from individuals generated from the non-diapause eggs after the nucleic acid introduction step. The method of producing said product.

7. A method for producing a genetically modified cloned silkworm, comprising the steps of: a step of removing the subesophageal ganglion from the female silkworm; a mating step of mating the female individuals after the removal step; a spawning induction step of contacting or introducing a muscarinic acetylcholine receptor agonist into the female individual after the mating step to induce spawning; an egg collection step of collecting fertilized eggs from the female individuals after the egg-laying induction step; 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 a female individual obtained from the fertilized eggs after the nucleic acid introduction step; a parthenogenetic induction step of subjecting the unfertilized eggs obtained in the unfertilized egg collection step to parthenogenetic induction treatment; and A selection step of selecting genetically modified organisms as genetically modified cloned silkworms from individuals generated after the parthenogenetic induction step. The method of producing said product.

8. The method according to claim 7 , wherein the silkworms used in the removal step are diapausing silkworms.

9. The method for producing an egg-laying egg, according to claim 7 or 8, wherein the muscarinic acetylcholine receptor agonist in the egg-laying induction step is selected from the group consisting of pilocarpine, cevimeline, carpronium, xanomeline, carbachol, bethanechol, aceclidine, albamelin maleate, and aclatonium napadisilate.

10. The method according to any one of claims 6 to 8, wherein the silkworms in the removal step are final stage larvae or pupae.

11. The method according to any one of claims 6 to 8, further comprising introducing a marker gene in the nucleic acid introduction step.

12. The method according to claim 11 , wherein the selection in the selection step is based on the expression of the marker gene.

13. The method according to any one of claims 6 to 8, wherein the parthenogenetic induction treatment is a high temperature treatment in which an unfertilized egg is exposed to 45°C to 50°C for 15 minutes to 20 minutes.

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