JP7664633B2 - Method for producing transgenic silkworms using diapause eggs - Google Patents

Description

The present invention relates to a method for producing genetically modified silkworms using diapause eggs.

Genetic engineering of living organisms is essential for analyzing gene functions and producing useful proteins. Traditionally, Escherichia coli and yeast have been used primarily as host organisms for genetic engineering. While these hosts have the advantage of being easy to culture and capable of mass-producing in a short period of time, they have the drawback of being difficult to mass-produce proteins from.

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 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 are being 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.

To produce transgenic silkworms, the 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 when 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.

By the way, most silkworms are univoltine, in which adults usually emerge once a year. Univoltine silkworms lay diapause eggs, which go into diapause. This diapause state is maintained even after microinjection, so even if the gene is introduced into diapause eggs, a transformant cannot be obtained. Therefore, non-diapause eggs that do not go into diapause are used in the microinjection method. However, most non-diapause strains that lay non-diapause eggs are experimental strains with poor silk productivity, and are unsuitable for industrial use. Therefore, in the past, it was necessary to 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.

To solve the above problems, methods have been developed to obtain dormant eggs or non-diapause eggs that do not enter a dormant state (often collectively referred to as "non-diapause eggs, etc." in this specification) from practical dormant strains. For example, there is a diapause breaking method that prevents dormant eggs from entering a dormant state by applying external physical or chemical stimuli and maintains embryo development, and a non-diapause egg laying method that treats parent silkworms of a dormant strain to cause them to lay non-diapause eggs.

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 subjected to a physical stimulus of microinjection in addition to the chemical stimulus of acid. From the results of Non-Patent Document 3, as Zhao et al. consider, the combined use of the diapause breaking method and the microinjection method is considered to be unsuitable for the production of genetically modified silkworms in this field because both methods impose excessive stress on the eggs.

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 microinjected to successfully 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 drops drastically, as with the acid immersion method.

As mentioned above, while conventional methods can prepare non-diapause eggs from diapause strain silkworms, there is a problem in that the hatching rate drops significantly after the subsequent microinjection process. Because genetic recombination only occurs in a portion of the eggs that have been 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 was needed that not only could obtain non-diapause eggs, but could also maintain the hatching rate after microinjection.

To solve the above problems, Patent Document 2 discloses a method of microinjecting non-diapause eggs obtained from a diapause strain of silkworms using a diapause hormone antibody. As disclosed in Non-Patent Document 6, the method using the diapause hormone antibody involves injecting a neutralizing antibody against the diapause hormone into parent silkworms at the final instar larva to early pupa stage, thereby functionally inhibiting the diapause hormone of the female parent silkworm, thereby causing the diapause strain of silkworms 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.

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.

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

Therefore, with conventional technology, the expression level and quality of the target gene differs for each line or individual transformant, resulting in problems such as instability in the quality and production volume of the protein produced. This is a particularly serious problem when using a mass production system using genetically modified silkworms to manufacture raw materials for human pharmaceuticals, which require high quality.

As described above, the production of genetically modified silkworms and the maintenance of their lineage require a great deal of time and effort, which in turn increases costs. Furthermore, even if such time and effort are spent, the genetically modified silkworms obtained are still subject to the problem of genetic instability.

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 minimal labor. At the same time, there is also a need for technologies to maintain the genetic stability of the transgenic silkworms that have been produced.

JP 2003-88274 A Patent No. 6765803

Tamura T., et al. (2000) Nat. Biotechnol.18, 81-84. Tamura, T. (2007) Development and use of methods for producing transgenic silkworms (Series: 21st Century Agriculture: Genetic Engineering Research of Animals and Microorganisms, edited by the Agricultural Science Society of Japan) pp57-76. Yokendo. Tokyo. Zhao AC, et al., 2012, Insect Science, 19: 172-182 Eiichi Ozegawa et al., 2000, Japanese Journal of Sericulture, 69(6): 369-375 Shimizu, Isamu, 1991, Ōdōkon, 35: 81-91 Shiomi K, et al., 1994, J. Insect Physiol., 40(9):693-699

The objective of this project is to develop and provide technology to produce practical strains of genetically modified silkworms suitable for industrial use in a relatively short period of time with little labor, and to develop and provide technology to genetically maintain the genetically modified silkworms that have been produced. This will reduce the costs required for producing genetically modified silkworms and maintaining the strains.

In order to solve the above problems, the present inventors conducted intensive research and found that when diapause-breaking of diapause eggs was performed using dimethyl sulfoxide (DMSO) and the resulting eggs were subjected to microinjection, a high hatching rate of 30% or more was obtained. It was also revealed that the desired genetically modified silkworms could be produced by subsequent selection. As explained in the above-mentioned Non-Patent Document 3, the combined use of the diapause-breaking method and the microinjection method was previously considered unsuitable for producing genetically modified silkworms because it damaged the eggs and caused 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 the eggs is achieved by immersing or coating the diapause eggs in a DMSO-containing solution containing the target chemical substance. As such, the combination of dormancy breaking and microinjection methods has been considered taboo in the field, and the above results were unexpected findings. The present invention is based on this new finding and provides the following:

(1) A method for producing genetically modified silkworms, comprising: a fertilized egg collection step 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 target nucleic acid 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 lineage; 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 genetically modified silkworms from the next generation silkworms after the nucleic acid introduction step.
(3) The method according to (1) or (2), wherein the time for contacting 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 minutes to 20 minutes.

The method for producing genetically modified silkworms of the present invention provides a technology 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.

FIG. 1 is a flow diagram of a method for producing a transgenic silkworm of the present invention.

1. Method for Producing Transgenic Silkworms 1-1. Overview The present invention relates to a method for producing transgenic silkworms. The production method of the present invention is characterized in that a target nucleic acid is introduced into diapause eggs that have been obtained by subjecting diapause eggs to a diapause-breaking treatment other than an acid soaking treatment, and thus avoiding diapause. According to the present invention, transgenic silkworms of practical strains suitable for industrial use or transgenic cloned silkworms can be produced in a relatively short period of time and with little effort.

1-2. Definitions The following terms used in this specification are defined below.
"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.

"Genetically modified silkworms" (transgenic silkworms) refer to genetically modified silkworms or their progeny produced using genetic engineering technology. In this specification, genetically modified silkworms refer to, but are not limited to, genetically modified silkworms obtained by introducing foreign DNA into silkworm eggs using the microinjection method.

"Cloned silkworms" refers to silkworm individuals that have the same genome composition and that form a genetically homogeneous population with the same gene composition. In general, cloned silkworms are silkworm populations obtained by inducing parthenogenesis in unfertilized eggs obtained from parthenogenetic strains of silkworms, as described below.

In this specification, "genetically modified cloned silkworms" refers to genetically modified cloned silkworms. In genetically modified cloned silkworms, the introduced foreign DNA is identical between individuals belonging to the 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.

In this specification, a "lineage" is a group of individuals that share a specific genetic trait within the same species, and is roughly the same concept as a "strain." In this specification, lineage also includes mutants with mutations in specific genes and varieties with common morphology or properties. In principle, the biological species covered in this specification is silkworms. Therefore, unless otherwise specified, lineage in this specification means a "silkworm lineage," and silkworm individuals belonging to each lineage are called "lineage silkworms." For example, the dormant lineage described below is a dormant lineage of silkworms, that is, a "dormant silkworm lineage," and silkworms that belong to this dormant silkworm lineage are called "dormant lineage silkworms." 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.

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

In this specification, "avoiding diapause" means that diapause eggs that have the potential to enter a dormant state do not enter a dormant state through artificial treatment, such as breaking the diapause described below, and continue to develop, or are able to continue to do so.

In this specification, "diapause (silkworm) strain" refers to a population of individuals that lay diapause eggs with respect to a specific genetic trait called egg dormancy. Silkworms are classified into univoltine strains, bivoltine strains, and multivoltine strains. Of these, many univoltine strains are diapause silkworm strains that lay diapause eggs. A "univoltine strain" is a strain in which adults emerge once a year when reared under natural conditions, a "bivoltine strain" is a strain in which adults emerge twice a year, and a "multivoltine strain" is a strain in which adults emerge multiple times a year. In general, many of the derived strains that have an ancestral strain in a temperate region where winter exists are univoltine strains. However, even diapause silkworm strains 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.

In this specification, "diapause eggs" refers to eggs obtained from diapause silkworms and that have the potential to transition to a diapause state. It does not matter whether or not they have actually transitioned to a diapause state. Usually, diapause eggs can be obtained by having adult female diapause silkworms lay eggs under general rearing conditions. The diapause eggs cease embryonic development at the embryo stage about two days after laying and enter a diapause state with low temperature resistance (Yanagunuma 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.

In this specification, "non-diapause (silkworm) strain" refers to an individual population that lays non-diapause eggs that do not enter a diapause state during the egg stage. Generally, many of the derived strains that have ancestral strains in subtropical or tropical regions where there is no winter are multivoltine silkworm strains that repeat multiple generations per year, and these are non-diapause silkworm strains that lay non-diapause eggs. Specific examples of non-diapause silkworm strains include, but are not limited to, Mysore, Nistari, Pure Mysore, Annan, and Ringetsu.

In this specification, "breaking diapause" refers to preventing diapause eggs from going into a dormant state by applying various artificial processes to the dormant eggs. As mentioned above, in dormant eggs laid by diapause strain silkworms, early embryo development usually stops at the embryo stage around two days after laying and the eggs go into a dormant state. However, by breaking diapause, the dormant eggs do not go into a dormant state and continue to develop.

In this specification, "non-diapause eggs" refers to silkworm eggs that do not have the potential to enter a diapause state. Examples include non-diapause eggs obtained from multivoltine strains, non-diapause eggs obtained from diapause silkworm strains by non-diapause egg-laying processing, 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 enter a diapause state.

In this specification, "non-diapause egg-laying treatment" refers to a specific treatment applied to individuals of a diapause silkworm lineage that normally lays diapause eggs, thereby causing them to lay non-diapause eggs. A specific example of the specific treatment is the low-temperature dark blue-inducing treatment described in the method disclosed in JP 2017-085958 A.

In this specification, "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 that have the property of being 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 include the PK1 line, the P14 line, and a hybrid of Camboge x Nihon 106.

A "marker gene" is a polynucleotide consisting of a base sequence that codes for a marker protein, also known as a selection marker.

"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 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 allow easy identification and selection of the transformant.

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

In this specification, 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, and the type of the pigment is not important. 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.

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.

In this specification, "exocrine protein" refers to a protein that is secreted outside the 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.

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 cell selection step (S0104), an unfertilized egg collection step (S0105), and a parthenogenetic induction step (S0106).

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. Of 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 parthenogenetic 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.

1-3-1. Fertilized egg collection step 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 their 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.

Hypoplastic fertilized eggs can be prepared by allowing mated adult females to lay eggs naturally according to known egg collection methods. For females used for egg collection, a diapausing silkworm strain that lays diapausing eggs can be used. In principle, eggs laid by mated adult females from a diapausing silkworm strain are diapausing fertilized eggs.

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.

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.

The dormant eggs used in this process 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 strain. Furthermore, the dormant eggs used are not only fertilized or unfertilized eggs of G0 silkworms, but also fertilized or unfertilized eggs of G1 silkworms.

As mentioned above, dormant eggs will enter a dormant state two or more days after spawning, and it will be difficult to break the dormancy thereafter. Therefore, it is desirable to carry out this process 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 process (S0105) described below, when eggs of a parthenogenetic silkworm strain are used, unfertilized eggs can be collected directly from the ovaries removed by dissection or the like without going through spawning. When protected at, for example, 15°C after parthenogenesis induction, the eggs will enter a dormant state after six or more days, so it is desirable to carry out this process on eggs less than 144 hours (6 days), within 126 hours, within 108 hours, within 90 hours, or within 72 hours after parthenogenesis induction.

Unless otherwise specified, the dormancy-breaking treatment in this process involves contact with dimethyl sulfoxide (DMSO).

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.

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.

Many of the eggs into which nucleic acid is introduced have avoided diapause through the diapause-breaking step (S0102) described above. In this step, it is desirable to introduce nucleic acid into multiple eggs after the diapause-breaking step (S0102).

In this process, the "nucleic acid of interest" refers to a nucleic acid to be introduced into the silkworm to produce the desired genetically modified silkworm. For example, DNA or RNA is included. 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 introducing a gene, the type of gene is not important. For example, a gene encoding a protein or a functional nucleic acid (such as an RNAi molecule) may be used. A desired gene that can impart 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 process described below. In this process, a peptide (including a protein such as an enzyme) and/or a low molecular weight compound can also 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 the introduction of the nucleic acid, after the introduction of the nucleic acid, or simultaneously with the introduction of the nucleic acid.

The gene introduced in this process may be of any biological species. 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 genetically modified silkworms, the foreign DNA may be an expression unit that includes an IgG antibody gene and gene expression regulatory regions such as a promoter and a terminator.

This step can be carried out by a method known in the art for introducing a foreign gene into silkworms. For example, if the expression vector is a plasmid having transposon inverted terminal repeats at both ends of the target DNA (Handler AM. et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 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.

Although not limited to this, the introduction of the desired nucleic acid into the egg is generally performed using a special injection device that utilizes air pressure. 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. There is no particular limit to the amount of nucleic acid to be introduced. It may be determined appropriately depending on the type, properties, and purpose of the nucleic acid. It is usually 1 nL to 5 nL.

After nucleic acid introduction, the eggs can be incubated under appropriate conditions, for example at 25°C, until they hatch.

The "recombinant selection step" (S0104) is a step of selecting genetically modified silkworms from the silkworms generated after the nucleic acid introduction step (S0103).

After the nucleic acid introduction process, G0 silkworms contain somatic cells and germ cells that have undergone genetic modification, but it is the genetic modification that has occurred in the germ cells that is passed on to the next generation. Therefore, this process is carried out on G1 silkworms.

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 no gene is introduced into the G1 fertilized eggs by microinjection, 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.

When using G1 unfertilized eggs obtained through the unfertilized egg collection step (S0105) from G0 silkworms of the parthenogenetic silkworm lineage after the nucleic acid introduction step (S0103), this step involves carrying out the parthenogenesis induction step (S0106) on the G1 unfertilized eggs, and then carrying out the 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.

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 possible to go through the unfertilized egg collection step (S0105) of collecting unfertilized eggs from G0 female individuals after the nucleic acid introduction step (S0103). The parthenogenesis induction step (S0106) and, if necessary, the diapause breaking step (S0102) can be performed on the obtained G1 unfertilized eggs before carrying out this step. In this case, the silkworms obtained after this step are genetically modified cloned silkworms.

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 target nucleic acid introduced in the nucleic acid introduction step (S0103). For example, when the target nucleic acid is a foreign gene, genomic DNA or mRNA may be prepared from silkworms that have emerged 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.

Alternatively, if 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 the 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 sight, touch, smell, hearing, and taste).

1-3-5. Unfertilized egg collection process The "unfertilized egg collection process" (S0105) is, in principle, a process for collecting unfertilized eggs from individuals of a parthenogenetic silkworm line. This process is carried out together with the parthenogenetic induction process (S0106) described below when a genetically modified cloned silkworm is produced using a parthenogenetic silkworm line as the parent line. This process is essential when producing a genetically modified cloned silkworm from a parthenogenetic silkworm line.

This process can also be carried out exceptionally when the G0 silkworms obtained through the fertilized egg collection process are not parthenogenetic silkworm strains, and when the G1 eggs obtained from the G0 silkworms can develop with a high probability through parthenogenetic induction treatment.

Therefore, when performing this step and the parthenogenetic 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 the fertilized egg collection step.

In this process, adult female individuals are used in which unfertilized eggs have developed to a state where they can develop parthenogenetically within the body, i.e., into mature unfertilized eggs.

There are no limitations on the method for collecting unfertilized eggs. For example, they can be collected by dissection or natural spawning.

Specifically, the method of collecting ovaries by dissection involves, for example, cutting open the abdomen or tail of a female adult with a scalpel or dissecting scissors, then applying pressure to the abdomen with fingers to remove the ovaries through the incision. The removed ovaries can be immersed in water to loosen the clumped oviducts. Next, the ovaries are placed on a fine-meshed 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 floating oviduct tissue, and the unfertilized eggs remaining at the bottom are collected. This procedure can be repeated several times to collect only the unfertilized eggs.

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 employed 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 in a low temperature of 0-10°C, preferably 5°C, and stored at that temperature for 1-2 days, with a maximum of 1-7 days, after which the female silkworm is transferred from the low temperature to room temperature (23-28°C), where it is provided with an egg-laying mat and allowed to begin laying eggs under dark conditions.

1-3-6. Parthenogenesis induction step The "parthenogenesis induction step" (S0106) is a step of inducing parthenogenesis in the unfertilized eggs obtained in the unfertilized egg collection step (S0105). This step is carried out in combination with the unfertilized egg collection step (S0105). Therefore, the silkworms obtained through the unfertilized egg collection step (S0105) and this step are cloned silkworms.

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 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 used. 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. It is preferable to then keep the unfertilized eggs at 12°C to 18°C for 2 to 6 days.

The dormant unfertilized eggs obtained after this process have been induced to undergo parthenogenesis. However, because they are dormant eggs, they will not hatch. By subjecting these dormant eggs to the diapause-breaking process (S0102) described above, they will avoid diapause and development will continue, eventually leading to hatching.

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.

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

In each experimental group, male and female moths of the above strains were mated with the same strain and then raised at 5°C for 1 to 3 days. When it was time to collect eggs, the female moths were transferred to an egg-laying mat and allowed to lay eggs. All eggs obtained in this way were diapause eggs. After egg collection, they were treated with DMSO.

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.

Next, nucleic acids were 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.

The hatched larvae in each experimental group were transferred to a plastic box containing artificial diet and raised at 25-27°C until they reached adulthood. G0 (0th generation after injection) adults obtained from each experimental group were mated with their siblings to obtain G1 (1st generation after injection) eggs. At this time, a group of about 300 eggs obtained from one female was considered as one moth group. G1 eggs were immersed in 6N HCl for 1 hour to break diapause, washed with water, and 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 introduced marker gene was detected by the ocelli, 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 where one or more genetically modified silkworms were obtained was defined as the recombinant moth area, and the ratio of recombinant moth areas to the total moth areas was calculated as the recombinant silkworm acquisition rate (recombination rate).

(result)
The results are shown in Table 1.

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

Claims (6)

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;
the nucleic acid introduction step of introducing a nucleic acid of interest into the eggs after the diapause breaking step by a microinjection method; and the recombinant selection step of selecting a genetically modified organism from the next generation silkworms after the nucleic acid introduction step. 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;
the nucleic acid introduction step of introducing a nucleic acid of interest into the eggs after the diapause breaking step by a microinjection method; and the recombinant selection step of selecting a genetically modified organism from the next generation silkworms after the nucleic acid introduction step. The method according to claim 1 or 2, wherein the time for contacting with dimethyl sulfoxide is 10 minutes to 1 hour. The method according to any one of claims 1 to 3, further comprising introducing a marker gene in the nucleic acid introduction step. The method of claim 4, wherein the selection in the recombinant selection step is based on the expression of the marker gene. The method for producing a plant according to claim 2 , or any one of claims 3 to 5 which cite 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 to 20 minutes.

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