Method for producing transgenic silkworms using diapause eggs

Abstract

To develop and provide a technique for making commercial lines of transgenic silkworms in a short time and with little effort.SOLUTION: A dormant egg is subjected to a dormancy breaking process with dimethyl sulfoxide (DMSO), and a resultant egg undergoes the introduction of a target nucleic acid, to make a transgenic silkworm.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Genetic engineering of living organisms is essential for analyzing gene functions and producing useful proteins. Traditionally, Escherichia coli and yeast have been used mainly as host organisms for genetic engineering. These hosts have the advantage of being easy to culture and capable of mass-producing in a short period of time, but they have the problem of being difficult to mass-produce proteins.

[0003] In recent years, the spotlight has been on silkworms (Bombyx mori) as a host organism suitable for mass production of proteins and for which genetic engineering technology has been established. Silkworms are insects that have long been used industrially to produce silk, and they are able to mass-produce silk thread from the cocoons made by their larvae in a short period of time. Taking advantage of this characteristic, it has become possible to mass-produce useful proteins other than silk thread using genetic engineering technology, and some of these have been marketed as animal testing drugs, etc. At present, development is underway to produce pharmaceutical ingredients for humans using genetically modified silkworms (transgenic silkworms) obtained through genetic engineering technology.

[0004] To produce transgenic silkworms, a microinjection method is used in which a desired gene is injected into eggs using a transposon (Non-Patent Document 1). In the microinjection method, donor DNA, a transferase derived from a transposon, and other recombination activity are introduced into eggs at the early stage of embryo development to produce transgenic silkworms. 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. The desired gene must be introduced into the egg within this period, specifically 2 to 8 hours after spawning. This is because the efficiency of emergence of transformants decreases significantly after this period during which embryo development progresses (Non-Patent Document 2). In other words, to produce transgenic silkworms using existing methods, it is important to introduce the target gene into eggs in which embryo development does not stop within a specific time after spawning.

[0005] 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. Therefore, non-diapause eggs that do not enter a state of diapause are used in the microinjection method. However, many of the non-diapause strains that lay non-diapause eggs are experimental strains with poor silk productivity, etc., and are not suitable for industrial use. Therefore, in the past, it was necessary to carry out genetic recombination by microinjection using non-diapause eggs obtained from a non-diapause strain, and then cross the obtained genetically modified silkworms with a diapause practical strain to develop a practical strain suitable for industrial use.

[0006] 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 line that is a diapause line. For example, there is a diapause breaking method in which the transition of diapause eggs to a diapause 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 diapause line are treated to cause them to lay non-diapause eggs.

[0007] Non-Patent Document 3 discloses a method of microinjection into diapause eggs that do not enter a diapause state, that is, diapause eggs that have avoided diapause, obtained by acid immersion, which is one of the methods for breaking diapause. Normally, acid immersion is a method of breaking diapause by immersing diapause eggs 24 hours after laying in a hydrochloric acid solution. In Non-Patent Document 3, genetic recombination is performed by microinjection on bivoltine diapause eggs that have been acid immersed within 3 hours after laying. However, this method has the problem that the hatching rate thereafter is significantly reduced to only 3.4 to 4.6%, because the eggs are given a physical stimulus of microinjection in addition to the chemical stimulus of acid. From the results of Non-Patent Document 3, as discussed by Zhao et al., the combined use of the diapause breaking method and the microinjection method in this field is considered to be unsuitable for the production of genetically modified silkworms because both methods give excessive stress to the eggs.

[0008] The low-temperature dark incubation method is a method in which parent eggs are incubated under low-temperature dark conditions, and the hatched parent silkworms are made to lay non-diapause eggs (Non-Patent Documents 4 and 5). For example, in Non-Patent Document 1, microinjection is performed on non-diapause eggs obtained from parent silkworms that have been subjected to low-temperature dark incubation to obtain genetically modified silkworms. In addition, in Patent Document 1, parent eggs are not only incubated under low-temperature dark incubation, but also parent larvae are reared under full light (24-hour light period) to cause them to lay non-diapause eggs, and the non-diapause eggs are then subjected to microinjection to obtain recombinant silkworms. 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). In addition, when microinjection is performed on non-diapause eggs obtained by this method, there is also the problem that the hatching rate is extremely reduced, as in the case of the acid immersion method.

[0009] As described above, although the conventional method can prepare non-diapause eggs from diapause strain silkworms, there is a problem that the hatching rate significantly decreases due to the subsequent microinjection treatment. Since genetic recombination occurs only in a portion of the eggs that are microinjected, if the hatching rate after microinjection is low, the success rate of producing genetically modified silkworms will inevitably be low. Therefore, in order to efficiently produce genetically modified silkworms, a technology that can not only obtain non-diapause eggs, but also maintain the hatching rate after microinjection is required.

[0010] In order to solve the above problems, Patent Document 2 discloses a method of microinjecting non-diapause eggs obtained from a diapause strain silkworm using a diapause hormone antibody. As disclosed in Non-Patent Document 6, the method using the diapause hormone antibody is a method in which a neutralizing antibody against the diapause hormone is injected into parent silkworms from the final instar larva to the early pupa stage, and the diapause hormone of the female parent silkworm is functionally inhibited, thereby causing the diapause strain silkworm to lay non-diapause eggs. As a result, Patent Document 2 discloses that the hatching rate after microinjection is high regardless of the silkworm strain. However, this method is also not easy in terms of labor, as it requires two injections in addition to the preparation of the antibody.

[0011] Furthermore, genetically modified silkworms obtained by conventional microinjection methods 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 derived from a transposon, the insertion of the target gene occurs at a random position on the genome, and 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, 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.

[0013] Therefore, in conventional techniques, the expression level and quality of the target gene differs for each line or individual of the transformant, resulting in the problem that the quality and production amount of the produced protein are not stable. This is a major problem when using a mass production system using transgenic 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 lineage require a great deal of time and effort, which increases costs. Furthermore, even if time and effort are spent, the transgenic silkworms obtained have the problem of being genetically unstable.

[0015] Therefore, there is a need for new technologies that can reduce the costs involved by producing transgenic silkworms in a short period of time with little labor, and at the same time, there is a need for technologies to maintain the genetic stability of the transgenic silkworms that have been produced. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] JP 2003-88274 A [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 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 [Non-Patent Document 6] Shiomi K, et al., 1994, J. Insect Physiol., 40(9):693-699 Summary of the Invention [Problem to be solved by the invention]

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

[0019] In order to solve the above problems, the present inventors have conducted intensive research and found that when diapause breaking of diapause eggs is performed using dimethyl sulfoxide (DMSO) and the resulting eggs are subjected to microinjection, a high hatching rate of 30% or more can be obtained. It has also been revealed that the desired genetically modified silkworms can be produced in the subsequent selection. As explained in the above-mentioned Non-Patent Document 3, the combined use of the diapause breaking method and the microinjection method has been considered unsuitable for producing genetically modified silkworms because it damages the eggs and gives excessive stress. In fact, JP 2012-044889 A also discloses a diapause breaking method and a chemical substance introduction method using DMSO, similar to the present invention, but states that "with regard to the microinjection method, it is difficult to introduce a chemical substance without damaging the eggshell because silkworm eggs have a thick and strong eggshell" (paragraph number 0006), and the introduction of a chemical substance into an egg is realized by immersing or coating the diapause eggs in a DMSO-containing solution containing the target chemical substance. Thus, the combination of the dormancy breaking method and the microinjection method has been considered taboo in the art, and the above results were unexpected findings. The present invention is based on the new findings and provides the following.

[0020] (1) A method for producing genetically modified silkworms, comprising: a fertilized egg collection step of collecting fertilized eggs from individuals of a dormant silkworm strain; a diapause breaking step of breaking the diapause of the fertilized eggs collected in the fertilized egg collection step by contacting them with dimethyl sulfoxide; a nucleic acid introduction step of introducing a nucleic acid of interest into the eggs after the diapause breaking step by microinjection; and a recombinant selection step of selecting genetically modified silkworms from the next generation silkworms after the nucleic acid introduction step. (2) A method for producing genetically modified silkworms, comprising: an unfertilized egg collection step of collecting unfertilized eggs from individuals of a parthenogenetic silkworm line; a parthenogenetic induction step of subjecting the unfertilized eggs collected in the unfertilized egg collection step to a parthenogenetic induction treatment; a diapause breaking step of breaking the diapause of the unfertilized eggs after the parthenogenetic induction step by contact treatment with dimethyl sulfoxide; a nucleic acid introduction step of introducing a nucleic acid of interest into the eggs after the diapause breaking step by microinjection; and a recombinant selection step of selecting a genetically modified silkworm from the next generation silkworms after the nucleic acid introduction step. (3) The method according to (1) or (2), wherein the time for the contact treatment with dimethyl sulfoxide is 10 minutes to 1 hour. (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 recombinant selection step is based on the expression of the marker gene. (6) The method according to any one of (2) to (5), 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

[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 labor. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is a flow diagram of a method for producing a transgenic silkworm of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] 1. Method for producing transgenic silkworms 1-1. Overview The present invention relates to a method for producing transgenic silkworms, which is characterized in that a nucleic acid of interest is introduced into diapause eggs obtained by subjecting diapause eggs to a diapause-breaking treatment other than an acid soaking treatment, thereby avoiding diapause. 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 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.

[0025] "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.

[0026] 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.

[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] 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.

[0029] 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.

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

[0031] 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. Specific examples of dormant silkworm strains include, but are not limited to, Daizo, J-137, Shi-146, J-603, J-604, Chu-604, Chu-605, Chu-514, Chu-515, Chu-9.0, J-9.0, Shunrei, Kanetsuki, Hitachi, Nishiki, J-502, Shi-146, Shi-122, Toku-Shi 2, Ou-7, Toku-Ou-4, and Kaku-Shina.

[0032] 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.

[0033] 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.

[0034] As used herein, "breaking diapause" refers to preventing diapause eggs from going into a diapause state by subjecting them to various artificial treatments. As described above, diapause eggs laid by diapause-resistant silkworms usually enter a diapause state around the second day after laying, when early embryo development stops at the embryo stage. However, by breaking diapause, diapause eggs do not enter a diapause state and continue to develop.

[0035] In this specification, "non-diapause eggs" refers to silkworm eggs that do not have the potential to become diapause. Examples include non-diapause eggs obtained from a multivoltine lineage, non-diapause eggs obtained from a diapause silkworm lineage by a 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 potential to become diapause.

[0036] In the present specification, the term "non-diapause egg-laying treatment" refers to a specific treatment that is performed on individuals of a diapause silkworm line that normally lays diapause eggs, to cause 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.

[0037] 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, many 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". Parthenogenetic silkworm lineages are not limited, but examples thereof include PK1 lineage, P14 lineage, and a hybrid of Camboge x Nihon 106.

[0038] A "marker gene" is a polynucleotide consisting of a base sequence that codes for a marker protein, also called a selection marker.

[0039] The term "labeled protein" refers to a protein that can confer new traits 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.

[0040] 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. The excitation wavelength and the fluorescence wavelength are not particularly limited. 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 1-3. Preparation method The flow of the method for producing a transgenic silkworm 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 diapause breaking step (S0102), a nucleic acid introduction step (S0103), a recombinant selection step (S0104), an unfertilized egg collection step (S0105), and a parthenogenetic induction step (S0106).

[0045] The method for producing a genetically modified silkworm of the present invention can be broadly divided into a method for producing a genetically modified silkworm using a fertilized egg and a method for producing a genetically modified silkworm using an unfertilized egg. Among the above steps, the fertilized egg collection step (S0101), the diapause breaking step (S0102), the nucleic acid introduction step (S0103), and the recombinant selection step (S0104) are essential steps in the method for producing a genetically modified silkworm using a fertilized egg, and the diapause breaking step (S0102), the nucleic acid introduction step (S0103), the recombinant selection step (S0104), the unfertilized egg collection step (S0105), and the parthenogenesis induction step (S0106) are essential steps in the method for producing a genetically modified silkworm using an unfertilized egg. Each step in the method for producing a genetically modified silkworm of the present invention will be specifically described below.

[0046] 1-3-1. Fertilized egg collection process The "fertilized egg collection step" (S0101) is a selection step for collecting dormant fertilized eggs to be used in the diapause breaking step (S0102). This step is performed to obtain fertilized eggs of G0 silkworms (nucleic acid introduced 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 introduced 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.

[0047] The dormant fertilized eggs can be prepared by naturally spawning female adults after mating according to a known egg collection method. A dormant silkworm strain that spawns dormant eggs can be used as the female individuals used for egg collection. In principle, eggs spawned by dormant silkworm strains and mated female adults are dormant fertilized eggs.

[0048] There is no limitation on the method of collecting eggs. Usually, a method is adopted in which an egg-laying mat is provided to a mated female adult and the adult female is allowed to lay eggs on the mat. When an egg-laying mat is provided to a mated female adult, egg-laying often begins within a few hours after the mating mat is provided. After this step, dormant fertilized eggs can be obtained for use in the subsequent dormancy breaking step.

[0049] 1-3-2. Dormant breaking process The "diapause breaking step" (S0102) is a step of subjecting diapause-breaking treatment to diapause eggs. The diapause-breaking treatment is artificially performed on diapause eggs, thereby preparing diapause-free eggs.

[0050] The dormant eggs used in this step may be either fertilized or unfertilized eggs. However, in the case of unfertilized eggs, the dormant eggs are those that have been subjected to the unfertilized egg collection step (S0105) and parthenogenesis induction step (S0106) described below from a parthenogenetic silkworm line. In addition, the dormant eggs used are not only fertilized or unfertilized eggs of G0 silkworms, but also fertilized or unfertilized eggs of G1 silkworms.

[0051] As described above, dormant eggs go into a dormant state two or more days after spawning, and it is difficult to break the dormancy thereafter. Therefore, this step is desirably performed on eggs less than 48 hours, within 42 hours, within 36 hours, within 30 hours, or within 24 hours after spawning. In addition, as described in the unfertilized egg collection step (S0105) described below, when eggs of a parthenogenetic silkworm strain are used, unfertilized eggs can be collected directly from ovaries removed by dissection or the like without going through spawning. When protected at, for example, 15°C after parthenogenetic induction, the eggs go into a dormant state after six days or more, so this step is desirably performed on eggs less than 144 hours (6 days), within 126 hours, within 108 hours, within 90 hours, or within 72 hours after parthenogenetic induction.

[0052] The dormancy-breaking treatment carried out in this step is a contact treatment with dimethyl sulfoxide (DMSO), unless otherwise specified.

[0053] In the present specification, "contact treatment with dimethyl sulfoxide (DMSO)" (often abbreviated as "DMSO treatment" in the present specification) refers to a method of giving a chemical stimulus to diapause eggs by contact treatment with a DMSO solution to prevent the transition to a dormant state. The concentration of the DMSO solution used is not limited, but is preferably 90% to 100%, 92% to 100%, 94% to 100%, 96% to 100%, or 98% to 100%. In the present specification, "contact" refers to the whole or part of an object being in contact with another object. Therefore, contact with a DMSO solution includes, for example, immersing a diapause egg in a DMSO solution, spraying or applying a DMSO solution to a diapause egg, and the like. The contact time with the DMSO solution is not limited. It may be within the range of 7.5 minutes to 1.5 hours, 10 minutes to 1 hour, 15 minutes to 55 minutes, 20 minutes to 50 minutes, 25 minutes to 45 minutes, or 30 minutes to 40 minutes. After DMSO treatment, it is preferable to thoroughly wash the diapause eggs with water, a buffer, or the like to remove DMSO. This process prevents diapause of the dormant eggs.

[0054] 1-3-3. Nucleic acid introduction step The "nucleic acid introduction step" (S0103) is a step of introducing a nucleic acid of interest into the eggs after the diapause breaking step (S0102). The eggs used in this step are fertilized or unfertilized eggs of G0 silkworms.

[0055] Many of the eggs into which the nucleic acid is to be introduced are in a state in which they have avoided diapause due to the diapause-breaking step (S0102). In this step, it is desirable to introduce the nucleic acid into a plurality of eggs after the diapause-breaking step (S0102).

[0056] In this step, the "nucleic acid of interest" refers to a nucleic acid to be introduced into a silkworm in order to produce a transgenic silkworm of interest. 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, 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. In this step, a peptide (including a protein such as an enzyme) and / or a low molecular weight compound may be introduced together with the nucleic acid as necessary. In this case, the order of introduction with the nucleic acid is not important. It may be before, after, or simultaneously with the introduction of the nucleic acid.

[0057] The origin of the gene introduced in this step does not matter. It may be derived from the host silkworm, or may be derived from another organism, such as a human. As an 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 a terminator.

[0058] 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.

[0059] The introduction of the target nucleic acid into the egg is generally performed using a special injection device that utilizes air pressure, although this is not limited thereto. For example, the method described in 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. It may be appropriately determined depending on the type, properties, and purpose of the nucleic acid. It is usually 1 nL to 5 nL.

[0060] After the introduction of the nucleic acid, the eggs may be incubated under appropriate conditions, for example, at 25° C., until they hatch.

[0061] 1-3-4. Recombinant selection process The "recombinant selection step" (S0104) is a step of selecting genetically modified silkworms from the silkworms generated after the nucleic acid introduction step (S0103).

[0062] The G0 silkworms that have undergone the nucleic acid introduction process contain somatic cells and germ cells in which genetic recombination has occurred, but it is the genetic recombination that has occurred in the germ cells that is passed on to the next generation. Therefore, this process is carried out using G1 silkworms.

[0063] When using G1 fertilized eggs obtained through the fertilized egg collection step (S0101) from G0 silkworms of a dormant silkworm strain after the nucleic acid introduction step (S0103), the G1 fertilized eggs are subjected to a diapause-breaking step (S0102) and then this step is carried out. The silkworms obtained after this step are normal genetically modified silkworms. Note that since gene introduction by microinjection is not performed on the G1 fertilized eggs, the diapause-breaking treatment for the G1 fertilized eggs is not limited to the DMSO treatment described above, and other known diapause-breaking methods can also be used. Examples include acid immersion treatment, centrifugation treatment, and oxygen treatment.

[0064] When using G1 unfertilized eggs obtained through an unfertilized egg collection step (S0105) from G0 silkworms of a parthenogenetic silkworm lineage after the nucleic acid introduction step (S0103), this step involves carrying out a parthenogenesis induction step (S0106) on the G1 unfertilized eggs, and then carrying out a diapause breaking step (S0102). The individuals that hatch after this step are cloned silkworms. This step is carried out on these hatched cloned silkworms. Therefore, the silkworms obtained after this step are genetically modified cloned silkworms.

[0065] Furthermore, as an exceptional case, even if the G0 silkworm has undergone the fertilized egg collection step, a high parthenogenesis rate and non-diapause rate may be expected in the G1 eggs collected after the nucleic acid introduction step (S0103) depending on the combination of parent silkworm strains used to produce the G0 silkworms by crossbreeding, etc. In such a case, even if the G0 silkworm has undergone the fertilized egg collection step (S0101), it is also possible to go through the unfertilized egg collection step (S0105) of collecting unfertilized eggs from the G0 female individual after the nucleic acid introduction step (S0103). The obtained G1 unfertilized eggs can be subjected to the parthenogenesis induction step (S0106) and, if necessary, the diapause breaking step (S0102), and then this step can be carried out. In this case, the silkworm obtained after this step is a genetically modified cloned silkworm.

[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 (S0103). 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 (S0103), 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 easily selected based on the expression of the marker gene. The marker protein produced by the 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 directly by detecting the activity of the marker protein itself, or indirectly through 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] 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 lineage. This step is carried out together with the parthenogenetic induction step (S0106) described below when a genetically modified cloned silkworm is produced using a parthenogenetic silkworm lineage as the parent lineage. This step is essential when producing a genetically modified cloned silkworm from a parthenogenetic silkworm lineage.

[0069] In addition, this process can be carried out exceptionally when the G0 silkworms obtained through the fertilized egg collection process are not a parthenogenetic silkworm strain, and when the G1 eggs obtained from the G0 silkworms can develop with a high probability through parthenogenetic induction treatment.

[0070] Therefore, when performing this step and the parthenogenesis induction step (S0106) described below, the dormant eggs used in the diapause breaking step (S0102) are, in principle, dormant unfertilized eggs obtained from individuals of a parthenogenetic silkworm lineage, but as an exception, as described above, they may be dormant unfertilized eggs or non-dormant unfertilized eggs obtained from individuals that have undergone a fertilized egg collection step.

[0071] In this step, adult female individuals in which unfertilized eggs have developed into a state in which parthenogenesis is possible within the body, that is, into mature unfertilized eggs, are used.

[0072] The method for collecting unfertilized eggs is not limited, and examples thereof include a method for collecting eggs by dissection or a method for collecting eggs by natural spawning.

[0073] Specifically, the method of collecting by dissection involves, for example, incising the abdomen or tail of a female adult with a scalpel or dissecting scissors, and then applying pressure to the abdomen with fingers to remove the ovaries from the incision. The aggregated oviducts 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 fingers to separate the oviduct tissue and unfertilized eggs. Finally, water is poured through the mesh to remove the oviduct tissue floating on the water, and the unfertilized eggs remaining at the bottom are collected. This operation can be repeated several times to collect only the unfertilized eggs.

[0074] Unfertilized eggs can also be collected by natural egg-laying, in which unmated female individuals are allowed to lay eggs. There are no limitations on the egg-collection method. For example, a method is adopted in which an egg-laying mat is provided to an emerged female individual and the individual is allowed to lay eggs on the mat. Specifically, the emerged female individual is placed at a low temperature of 0 to 10°C, preferably 5°C, and stored at that temperature for 1 to 2 days, and at most 1 to 7 days, and then the female silkworm is transferred from the low temperature to room temperature (23 to 28°C), where an egg-laying mat is provided and the individual is allowed to start laying eggs under dark conditions.

[0075] 1-3-6. Parthenogenesis induction process The "parthenogenesis induction step" (S0106) is a step of performing parthenogenesis induction treatment on the unfertilized eggs obtained in the unfertilized egg collection step (S0105). This step is performed in conjunction with the unfertilized egg collection step (S0105). Therefore, the silkworms obtained through the unfertilized egg collection step (S0105) and this step are cloned silkworms.

[0076] 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. (Sugai Etsuji et al., 1983, Journal of Japanese Sericultural Science, Vol. 52, No. 1: 51-56), Hirokawa (Hirokawa Masahiko, 1990, Fukushima Sericultural Experimental Research Report, 24: 1-6), and Kosegawa et al. (Kosegawa E., 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 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.

[0077] 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 aforementioned diapause breaking process (S0102), they will avoid diapause and continue to develop, eventually leading to hatching. EXAMPLES

[0078] <Example 1: Production of transgenic silkworms using diapause eggs> (the purpose) We will verify hatching after microinjection using the method for producing genetically modified silkworms of the present invention, and the acquisition of genetically modified silkworms.

[0079] (method) In each experimental plot, the diapausing silkworm strain “Ariake” was used in two experimental plots (experimental plots A and B), the bivoltine strain “Nichi 137” was used in one experimental plot (experimental plot C), and the bivoltine strain “Shi 146” was used in one experimental plot (experimental plot D). These strains were obtained from the National Agriculture and Food Research Organization of Japan.

[0080] In each experimental group, the male and female moths of the above strains were mated in the same strain and then raised at 5°C for 1 to 3 days. When collecting eggs, the female moths were transferred to an egg-laying mat and allowed to lay eggs. All eggs obtained here were diapause eggs. After egg collection, DMSO treatment was performed.

[0081] DMSO treatment was performed by placing eggs collected from each experimental group in a Petri dish and immersing them in 100% DMSO (Fujifilm Wako Pure Chemicals). The DMSO treatment time was 45 minutes for experimental groups A and B, and 15 minutes for experimental groups C and D, in an air incubator at 25°C. After treatment, the eggs were washed with water and then air-dried.

[0082] Next, nucleic acid was introduced into the DMSO-treated eggs by microinjection. In experimental groups A, C, and D, the helper plasmid pHA3PIG (Tamura T., et al., 2000, Nat. Biotechnol.18: 81-84) and the vector plasmid pBac [3xP3-DsRedafm] (Horn and Wimmer, 2000, Dev. Genes Evol., 210(12): 630-637) were introduced into the DMSO-treated eggs 4 to 6 hours after oviposition, and in experimental group B, pHA3PIG and the vector plasmid pBac [3xP3-EGFPafm] (Horn and Wimmer, 2000) were introduced into the DMSO-treated eggs 4 to 6 hours after oviposition. After injection, the eggs were incubated in a humidified plastic box at 25°C until they hatched. The hatching rate was calculated as the ratio of hatched eggs to the number of injected eggs.

[0083] The hatched larvae in each experimental group were transferred to a plastic box containing artificial diet and raised at 25-27°C until they became adults. G0 (0th generation after injection) adults obtained in each experimental group were mated with their siblings to obtain G1 (1st generation after injection) eggs. At this time, a collection of about 300 eggs obtained from one female was designated as one moth group. G1 eggs were immersed in 6N HCl for 1 hour to break diapause, washed with water, and then incubated in a moist box at 25°C to allow development to proceed. When each egg was irradiated with excitation light, if the fluorescence of the marker protein (DsRed or GFP) based on the marker gene introduced by the ocelli was detected, the egg that emitted fluorescence was transferred to a Petri dish. The larvae that hatched from the transferred eggs were raised to establish transgenic silkworms. The moth area in which one or more transgenic silkworms were obtained was designated as the transgenic moth area, and the ratio of the transgenic moth areas to the total moth areas was calculated as the transgenic silkworm acquisition rate (recombination rate).

[0084] (result) The results are shown in Table 1.

[0085] [Table 1]

[0086] From the above results, when diapause was broken by DMSO treatment, a hatching rate of 14.4 to 62.7% was obtained even after microinjection, regardless of the line, the type of gene introduced, or the DMSO treatment time. When eggs that had been treated to break diapause by acid immersion were microinjected, the hatching rate was only 3.4 to 4.6%, as reported by Zhao AC et al. (Non-Patent Document 3). This was an unexpected result, as compared to the fact that the present invention achieved a hatching rate that was 3 to 10 times higher. Furthermore, the emergence of transgenic silkworms was confirmed for each moth group for hatched individuals, and transgenic silkworms were obtained in approximately 20 to 40% of the moth groups in all experimental groups.

Claims

1. A method for producing a transgenic silkworm, comprising the steps of: a step of collecting fertilized eggs from individuals of the dormant silkworm strain; a diapause breaking step in which the fertilized eggs collected in the fertilized egg collecting step are subjected to a contact treatment with dimethyl sulfoxide to break the diapause; a nucleic acid introduction step of introducing a nucleic acid of interest into the eggs after the diapause breaking step by a microinjection method; and A step of selecting recombinants from the next generation of silkworms after the nucleic acid introduction step. The method of producing said

2. A method for producing a transgenic silkworm, comprising the steps of: an unfertilized egg collection step of collecting unfertilized eggs from individuals of the parthenogenetic silkworm strain; a parthenogenetic induction step of subjecting the unfertilized eggs collected in the unfertilized egg collection step to parthenogenetic induction treatment; a dormancy breaking step of breaking dormancy of the unfertilized eggs after the parthenogenetic induction step by contact treatment with dimethyl sulfoxide; a nucleic acid introduction step of introducing a nucleic acid of interest into the eggs after the diapause breaking step by a microinjection method; and A step of selecting recombinants from the next generation of silkworms after the nucleic acid introduction step. The method of producing said

3. 3. The method according to claim 1, wherein the contact time with dimethyl sulfoxide is from 10 minutes to 1 hour.

4. The method according to any one of claims 1 to 3, further comprising introducing a marker gene in the nucleic acid introduction step.

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

6. The method according to claim 2, or any one of claims 3 to 5 which cites claim 2, 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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