JP2023119400A - Method for making transgenic silkworm using dormant egg - Google Patents

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

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

Gene recombination technology for organisms is essential for functional analysis of genes and production of useful proteins. Conventionally, Escherichia coli and yeast have mainly been used as host organisms for gene recombination. These hosts have the advantage of being easy to culture and capable of mass growth in a short period of time, but have the problem of difficulty in mass production of proteins.

Therefore, in recent years, the silkworm (Bombyx mori) has been in the spotlight as a host organism suitable for mass production of proteins and for which genetic recombination technology has been established. Silkworms are insects that have been industrially used for the production of silk since ancient times, and can mass-produce silk thread from cocoons made by larvae in a short period of time. Using this property, it has become possible to mass-produce useful proteins other than silk thread by gene recombination technology, and some of them are marketed as animal test reagents. At present, the development of human drug substance production technology using genetically modified silkworms (transgenic silkworms) obtained by gene recombination technology is underway.

A microinjection method, in which a desired gene is injected into an egg using a transposon, is employed for the production of transgenic silkworms (Non-Patent Document 1). In the microinjection method, genetically modified silkworms are produced by introducing donor DNA, transferase derived from transposons, other recombination activities, etc. into eggs at the early stage of embryogenesis. Silkworm eggs are fertilized within 2 hours after spawning, after which naked nuclei without cell membranes called syncytia migrate to the surface of the eggs while repeatedly dividing. The desired gene must be introduced into eggs within this period, specifically 2 to 8 hours after being laid. This is because the emergence efficiency of transformants is significantly reduced after this period during which embryonic development progresses (Non-Patent Document 2). In other words, in order to produce genetically modified silkworms by existing methods, it is important to introduce a target gene within a specific time after spawning into eggs in which embryonic development does not stop.

By the way, most of the silkworms are monophyletic strains in which adults usually emerge once a year. The silkworms of the monomorphic line lay diapause eggs and become diapause in the eggs. Since this diapause state is maintained even after microinjection, it is impossible to obtain a transformant even if the gene is introduced into diapause eggs. Therefore, the microinjection method uses non-diapausing eggs that do not enter a dormant state. However, many of the non-diapause lines that lay non-diapause eggs are experimental lines with poor silk productivity and are not suitable for industrial use. Therefore, conventionally, non-diapause eggs obtained from non-diapause strains are used for genetic recombination by microinjection, and then the resulting genetically modified silkworms are crossed with dormant practical strains for industrial use. It was necessary to develop a practical system suitable for

In order to solve the above problems, dormant eggs or non-diapausing eggs that do not enter a dormant state (in this specification, these are often collectively abbreviated as "non-diapausing eggs, etc.") are selected from practical strains that are dormant strains. A method has been developed to obtain For example, a dormancy breaking method that avoids the transition of diapause eggs to a diapause state by external physical or chemical stimulation and maintains embryonic development, and a dormancy-strain parent silkworm is treated to lay non-diapause eggs. There is a non-dormant egg laying method.

Non-Patent Document 3 discloses a method of microinjecting dormant eggs that have not entered a dormant state, that is, dormant eggs that have avoided dormancy, obtained by acid-immersion treatment, which is one of the dormancy breaking methods. Acid pickling is usually a method of breaking dormancy by immersing dormant eggs 24 hours after laying in a hydrochloric acid solution. In Non-Patent Document 3, gene recombination is performed by a microinjection method on dormant eggs of a dimorphic line that have been acid-immersed within 3 hours after spawning. However, in this method, the eggs are subjected to a physical stimulus called microinjection in addition to the chemical stimulus of acid. From the results of Non-Patent Document 3, as Zhao et al. consider, in the field, both the dormancy breaking method and the microinjection method in combination give excessive stress to the eggs. considered unsuitable.

The low-temperature dark hypnosis method is a method in which parent eggs are hypnotized under low-temperature dark conditions, and non-diapause eggs are laid by the emerged parent silkworms (Non-Patent Documents 4 and 5). For example, in Non-Patent Document 1, genetically modified silkworms are obtained by microinjection into non-diapause eggs obtained from parent silkworms that have been subjected to low-temperature dark hypnotic treatment. In addition, in Patent Document 1, not only is the parent egg subjected to low-temperature dark hypnotic treatment, but also the parent larvae are reared under full light (24-hour light period) to lay non-diapause eggs, and microinjection into the non-diapause eggs. We have succeeded in obtaining recombinant silkworms by performing However, the low-temperature dark hypnotic method has the problem that the number of eggs laid by non-diapause eggs differs significantly depending on the silkworm strain, and sometimes non-diapause eggs are not laid at all (Patent Document 1). In addition, when the non-diapause eggs obtained by this method are subjected to microinjection, there is also a problem that the hatchability is extremely lowered as in the case of the acid pickling method.

As described above, although the conventional method can prepare non-diapause eggs and the like from diapause silkworms, there is a problem that the subsequent microinjection treatment significantly lowers the hatchability. Since genetic recombination occurs only in some microinjected eggs, if the hatching rate after microinjection is low, the success rate of producing genetically engineered silkworms is inevitably low. Therefore, in order to efficiently produce genetically modified silkworms, there has been a need for a technique that not only obtains non-diapause eggs, but also maintains the hatchability after microinjection.

In order to solve the above problem, Patent Document 2 discloses a method of microinjecting non-diapause eggs obtained from diapause silkworms using diapause hormone antibodies. In the method using dormancy hormone antibody, as disclosed in Non-Patent Document 6, a neutralizing antibody against dormancy hormone is injected into parent silkworms from last instar larvae to early pupae, and the dormancy hormone of the female parent silkworm is functionally inhibited. In this method, non-diapause eggs are laid in diapause strain silkworms by inhibiting diapause. As a result, the method of Patent Document 2 discloses that the hatching rate after microinjection is high regardless of the silkworm strain. However, this method also required preparation of the antibody and two injections, and was labor intensive, so it was difficult to say that it was simple.

Furthermore, the genetically modified silkworms obtained by the conventional microinjection method have the problem that the genome composition differs depending on the transformant, even if the introduced target gene is the same. This is because when genetic recombination is performed using the transposon-derived transferase activity, the insertion of the target gene occurs at random positions on the genome, and silkworm strains are maintained as a genetically heterogeneous population by mating. This is because

In addition, silkworms are generally stored in the egg state, and the maximum storage period is one year. Therefore, in order to maintain the lineage, it is necessary to breed them every year and breed them to obtain the eggs of the next generation. Repeated mating over a long period of time makes it difficult to ensure genetic stability even within the same strain.

Therefore, in the conventional techniques, the expression level and quality of the target gene differ from one strain to another or each individual transformant, and as a result, the quality and yield of the produced protein are unstable. . This is especially a big problem when producing raw materials for human medicines, which require high quality, in a mass production system using genetically modified silkworms.

As described above, the production of genetically modified silkworms and the maintenance of their strains require a great deal of time and effort, and the cost also increases accordingly. Furthermore, the genetically modified silkworm obtained by spending time and effort has the problem of genetic instability.

Therefore, there is a need for a new technology that can reduce the costs incurred by producing genetically modified silkworms in a short period of time with less labor. At the same time, there is also a need for techniques for genetically stably maintaining the produced genetically modified silkworms.

JP-A-2003-88274 Patent No. 6765803

Tamura T., et al. (2000) Nat. Biotechnol.18, 81-84. Tamura, Toshiki (2007) Development and utilization of genetically modified silkworm production method (Series 21st Century Agriculture: Genetic Engineering Research of Animals and Microorganisms, Agricultural Society of Japan) pp57-76. Yokendo. Tokyo. Zhao AC, et al., 2012, Insect Science, 19: 172-182 Eiichi Kosegawa, et al., 2000, Japanese Journal of Sericulture, 69(6): 369-375 Shimizu I., 1991, Odo Kun, 35: 81-91 Shiomi K, et al., 1994, J. Insect Physiol., 40(9):693-699

To develop and provide a technique for producing genetically modified silkworms of practical strains suitable for industrial use in a relatively short period of time with little labor, and to genetically maintain the produced genetically modified silkworms in a stable manner. It is to develop and provide technology. By doing so, the objective is to reduce the costs required for producing genetically modified silkworms and maintaining strains.

In order to solve the above problems, the present inventors conducted intensive research and found that when dormancy was broken using dimethyl sulfoxide (DMSO) in dormant eggs and microinjection was performed on the resulting eggs, 30% It was found that a higher hatching rate than above can be obtained. It was also revealed that the desired transgenic silkworms could be produced in the subsequent selection. As explained in Non-Patent Document 3 above, conventionally, the combination of the dormancy breaking method and the microinjection method has been considered unsuitable for the production of genetically modified silkworms because it damages the eggs and gives excessive stress. . In fact, Japanese Patent Application Laid-Open No. 2012-044889 also discloses a method for breaking dormancy and introducing a chemical substance using DMSO as in the present invention. , it is difficult to introduce chemical substances without damaging the eggshell” (Paragraph No. 0006). This is achieved by coating. As described above, the combination of the dormancy breaking method and the microinjection method was contraindicated in the relevant field, so the above results were unexpected findings. The present invention is based on this new finding, and provides the following.

(1) A method for producing a genetically modified silkworm, comprising a fertilized egg collection step of collecting fertilized eggs from individuals of a dormant silkworm strain, and contacting the fertilized eggs collected in the fertilized egg collection step with dimethyl sulfoxide. A dormancy breaking step for breaking dormancy, a nucleic acid introduction step for introducing a target nucleic acid into eggs after the dormancy breaking step by a microinjection method, and a group for selecting genetically modified organisms from next-generation silkworms after the nucleic acid introduction step The production method, comprising a transformant selection step.
(2) A method for producing a genetically modified silkworm, comprising a step of collecting unfertilized eggs from an individual of a parthenogenic silkworm strain, and parthenogenesis in the unfertilized eggs collected in the step of collecting unfertilized eggs. a dormancy-breaking step of breaking dormancy by contacting unfertilized eggs after the parthenogenesis-inducing step with dimethyl sulfoxide; The production method, comprising a nucleic acid introduction step of introducing a nucleic acid of interest, and a recombinant selection step of selecting genetically modified organisms from next-generation silkworms after the nucleic acid introduction step.
(3) The production method according to (1) or (2), wherein the contact treatment time with the dimethylsulfoxide is 10 minutes to 1 hour.
(4) The production method according to any one of (1) to (3), wherein a marker gene is further introduced in the nucleic acid introduction step.
(5) The production method according to (4), wherein the selection in the recombinant selection step is based on the expression of the marker gene.
(6) The production method according to any one of (2) to (5), wherein the parthenogenetic induction treatment is a high-temperature treatment in which the unfertilized egg is exposed to 45° C. to 50° C. for 15 to 20 minutes.

According to the method for producing genetically modified silkworms of the present invention, it provides a technique for producing practical strains of genetically modified silkworms suitable for industrial use or genetically modified cloned silkworms in a relatively short period of time with less labor. be able to.

FIG. 1 is a flow diagram of a method for producing a genetically modified 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 by introducing a nucleic acid of interest into a dormant egg that has avoided dormancy, which is obtained by subjecting the dormant egg to a dormancy breaking treatment other than the acid-immersion treatment. INDUSTRIAL APPLICABILITY According to the present invention, practical strains of genetically modified silkworms suitable for industrial use or genetically modified cloned silkworms can be produced in a relatively short period of time with less labor.

1-2. Definitions The following terms used in this specification are defined.
"Genetic recombination" refers to artificial modification of the natural genetic information of a host organism. Modification of genetic information as used herein includes addition, deletion, substitution, and the like of genetic information. Artificial modification of genetic information can be achieved by existing gene recombination techniques. For example, a method of adding genetic information not possessed by the host organism using a vector such as a plasmid or a transposon, or a method of destroying the genetic information possessed by the host organism. In addition, in the present specification, genome editing techniques for modifying the genome information of host organisms by genome editing are also included as gene recombination in a broad sense.

“Genetically modified silkworm” (transgenic silkworm) refers to a genetically modified silkworm produced using genetic recombination technology or its progeny. The genetically modified silkworm of the present specification refers to, but is not limited to, a genetically modified organism obtained by introducing foreign DNA into a silkworm egg by a microinjection method.

“Cloned silkworm” refers to silkworm individuals that have the same genomic composition and constitute a genetically homogeneous population with the same genetic composition. In general, silkworm populations obtained by subjecting unfertilized eggs obtained from parthenogenetic line silkworms, which will be described later, to parthenogenesis-inducing treatment, correspond to clone silkworms.

As used herein, the term “genetically modified cloned silkworm” refers to a genetically modified cloned silkworm. In transgenic cloned silkworms, the introduced foreign DNA is also identical among individuals belonging to the cloned population. Therefore, in principle, the quality and expression level of proteins encoded by foreign DNAs produced by individual transgenic cloned silkworms are the same.

As used herein, the term "strain" refers to a population of individuals having specific common genetic traits within the same species, and is almost the same concept as "strain". Mutant groups having mutations in specific genes, and varieties having common morphology or properties are also included in the strains herein. In principle, the target species of the present specification is the silkworm. Therefore, unless otherwise specified, the strains in the present specification refer to "silkworm strains", and silkworm individuals belonging to each strain are referred to as "strain silkworms". For example, the dormant strain described later is a dormant strain of silkworms, that is, a “diapausing silkworm strain”, and silkworms belonging to this dormant silkworm strain are referred to as “diapausing strain silkworms”. Note that when focusing on different genetic traits, one individual can belong to a plurality of strains. For example, dormant parthenogenetic strains have two genetic traits: dormancy and parthenogenesis. to belong to.

As used herein, the term "diapause (state)" refers to a state in which an organism temporarily suspends development, growth, or activity at a specific time in its life cycle and enters a dormant state.

In the present specification, "avoiding dormancy (state)" means that a dormant egg that originally had the potential to enter a dormant state continues to develop without entering a dormant state due to an artificial treatment such as breaking dormancy described later. To do or to continue.

As used herein, the term "diapause (silkworm) lineage" refers to a population of individuals that lay diapause eggs with respect to the specific genetic trait of egg diapause. Silkworms include unipotent, dimorphic, and polymorphic strains. Among them, most of the unimorphic strains become dormant silkworm strains that lay diapause eggs. A “monomerous strain” is a strain that produces adults once a year when reared under natural conditions, and a “bimorphic strain” is a strain that produces adults twice a year. A "pluripotent line" is a line in which adults occur multiple times a year. Many of the descendant strains that have ancestors in the temperate zone, where winters generally exist, are monomorphic strains. However, even diapause silkworm strains may lay non-diapause eggs by the non-diapause egg-laying treatment described later. Specific examples of dormant silkworm strains include, but are not limited to, Taizo, Nichi No. 137, Nichi No. 146, Nichi No. 603, Nichi No. 604, Naka No. 604, Naka No. 605, Naka No. 514, and Naka No. 515. , Chu 9.0, Sun 9.0, Shunrei, Shogetsu, Hitachi, Nishiki, Sun 502, Branch 146, Branch 122, Tokushi 2, Europe 7, Toku Europe 4, and Kakushina, etc. .

As used herein, the term "diapause egg" refers to an egg obtained from a diapause silkworm strain and having the potential to transition to a diapause state. It does not matter whether or not it actually transitions to the dormant state. Normally, diapause eggs can be obtained by causing female adults of diapause strain silkworms to lay eggs under general breeding conditions. Two days after spawning, the diapause egg stops embryonic development at the embryonic stage and enters a diapause state with cold tolerance (Toshinobu Yaginuma, 2015, Silk and Insect Biotech, 84: 100). This is considered to be one of the life cycle control phenomena based on the environmental response acquired by the silkworm for biennial. Therefore, many of the derived strains that have ancestors in temperate regions where winters generally exist are dormant strains.

As used herein, the term “non-diapause (silkworm) strain” refers to a population of individuals that lay non-diapause eggs that do not enter dormancy during the egg stage. Many of the derived strains that have ancestral strains in subtropical or tropical regions where winter does not generally exist are pluripotent silkworm strains that repeat multiple generations per year, and these become non-diapause silkworm strains that lay non-diapause eggs. Specific examples of non-dormant silkworm strains include, but are not limited to, Mysore, Nistali, Pure Mysore, Annan, and Wangetsu.

As used herein, the term "breaking dormancy" refers to avoiding transition of dormant eggs to a dormant state by subjecting dormant eggs to various artificial treatments. As described above, the diapause eggs laid by the diapause strain silkworm usually stop in early embryogenesis at the embryonic stage and enter a diapause state around the second day after oviposition. However, by breaking dormancy, dormant eggs continue to develop without becoming dormant.

As used herein, the term "non-diapause eggs" refers to silkworm eggs that do not have the potential to enter a diapause state. Non-diapause eggs obtained from pluripotent strains, non-diapause eggs obtained from diapause silkworm strains by non-diapause egg-laying treatment, and non-diapause eggs obtained by the method described in Japanese Patent No. 6765803 are exemplified. In the present specification, dormant eggs that have avoided dormancy by breaking dormancy are distinguished from non-dormant eggs because they have the potential to become dormant.

As used herein, the term “non-diapause egg-laying treatment” refers to subjecting an individual of the dormant silkworm line, which originally lays diapause eggs, to a specific treatment so as to lay non-diapause eggs. Specific examples of the specific treatment include low-temperature dark blue treatment described in the method described in JP-A-2017-085958.

As used herein, the term “parthenogenetic (silkworm) strain” refers to a strain in which parthenogenesis is induced with high efficiency. In parthenogenetic silkworm strains, parthenogenesis is induced by giving physical or chemical stimuli to unfertilized eggs. In general, many of the silkworm strains that are capable of parthenogenesis are dormant silkworm strains. Therefore, parthenogenic silkworm strains are also referred to herein as “dormant parthenogenic (silkworm) strains”. Parthenogenic silkworm strains are not limited, but include, for example, PK1 strains, P14 strains, and hybrids of Camboju x Hino 106.

A "marker gene" is a polynucleotide consisting of a base sequence encoding a marker protein, also called a selectable marker.

The term “marker protein” refers to a protein capable of conferring a new trait not found in the host silkworm by expressing a marker gene. The type of labeling protein is not particularly limited as long as its activity can be detected by a method known in the art. A labeling protein that is less invasive to transformants is preferred for detection. Examples thereof include fluorescent proteins, chromosynthetic proteins, luminescent proteins, exosecretory proteins, proteins that control external morphology, and the like. Fluorescent proteins, chromosynthetic proteins, luminescent proteins, and exocrine proteins are visually detectable under certain conditions without altering the external morphology of the transformants, making them very less invasive to transformants. Also, it is particularly suitable because it facilitates identification and selection of transformants.

As used herein, "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. Also, the excitation wavelength and 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), and the like. be done.

As used herein, a “pigment synthesis protein” is a protein involved in pigment biosynthesis, usually an enzyme. The term “pigment” as used herein refers to a low-molecular-weight compound or peptide capable of imparting a pigment to a transformant, regardless of the type thereof. Preferred are pigments that appear as the external color of the solid. Examples include melanin pigments (including dopamine melanin), ommochrome pigments, and pteridine pigments.

As used herein, the term "photoprotein" refers to a substrate protein capable of emitting light without requiring excitation light or an enzyme that catalyzes the luminescence of the substrate protein. Examples include luciferin or aequorin as a substrate protein and luciferase as an enzyme.

As used herein, the term “exocrine protein” refers to a protein that is secreted outside the cell or body, and includes exocrine enzymes, fiber proteins such as fibroin, and sericin. Exocrine enzymes include enzymes such as blasticidin that contribute to the degradation or inactivation of drugs and impart drug resistance to the host, as well as digestive enzymes.

1-3. Production Method The flow of the production method of the genetically modified silkworm of the present invention is shown in FIG. As shown in this figure, the production method of the present invention includes a fertilized egg collection step (S0101), a dormancy breaking step (S0102), a nucleic acid introduction step (S0103), a recombinant selection step (S0104), and an unfertilized egg collection step ( S0105), and a parthenogenesis induction step (S0106).

The method for producing a genetically modified silkworm of the present invention can be roughly divided into a method for producing a genetically modified silkworm using fertilized eggs and a method for producing a genetically modified silkworm using unfertilized eggs. Among the above steps, the fertilized egg collection step (S0101), the dormancy breaking step (S0102), the nucleic acid introduction step (S0103), and the recombinant selection step (S0104) are the methods for producing genetically modified silkworms using fertilized eggs. In addition, the dormancy 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 This is an essential step in the method for producing genetically modified silkworms using unfertilized eggs. 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 used in the dormancy breaking step (S0102). This step is performed to obtain fertilized eggs of G0 silkworms (nucleic acid-introduced generation) and fertilized eggs of G1 silkworms (first generation after nucleic acid-introduced generation). The transgenic silkworms obtained in this case are normal transgenic silkworms that are 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 later. The genetically modified silkworm obtained in this case is a genetically modified clone silkworm.

Dormant fertilized eggs may be prepared by allowing the female adult individuals after mating to naturally lay eggs according to a known egg collection method. A dormant silkworm strain that lays dormant eggs may be used for the female individuals used for egg collection. In principle, the eggs of the dormant silkworm strain and laid by the female adult individuals after mating are dormant fertilized eggs.

There is no limitation on the egg collection method. Usually, a method is adopted in which an egg-laying mount is given to a mated female adult individual, and eggs are laid on the mount to collect eggs. When an egg-laying mat is given to a mated female adult individual, egg-laying often starts within several hours after the application.
After this step, dormant fertilized eggs to be used in the next step of breaking dormancy can be obtained.

1-3-2. Dormancy Breaking Step The “dormancy breaking step” (S0102) is a step of subjecting dormant eggs to a dormancy breaking treatment. A dormant egg is artificially subjected to a dormancy breaking treatment to prepare a dormant egg avoiding dormancy.

The dormant egg used in this step may be either a fertilized egg or an unfertilized egg. However, in the case of unfertilized eggs, dormant eggs that have undergone the unfertilized egg collection step (S0105) and the parthenogenesis induction step (S0106) described later from the parthenogenic silkworm strain. Moreover, the diapause eggs to be used are not only G0 silkworm fertilized or unfertilized eggs, but also G1 silkworm fertilized or unfertilized eggs.

As mentioned above, diapause eggs transition to a diapause state after 2 days or more after spawning, and it becomes difficult to break diapause after that. Therefore, it is desirable to perform this step on eggs within 48 hours, within 42 hours, within 36 hours, within 30 hours, or within 24 hours after spawning. In addition, as described in the step of collecting unfertilized eggs (S0105) described later, when eggs of parthenogenic silkworm strains are used, unfertilized eggs can be collected directly from the ovary removed by dissection or the like without going through egg laying. After induction of parthenogenesis, for example, when protected at 15°C, the cells enter a dormant state after 6 days or more. Ideally, eggs should be within 1 hour, 90 hours, or 72 hours.

The dormancy breaking treatment performed in this step is contact treatment with dimethyl sulfoxide (DMSO) unless otherwise specified.

As used herein, "contact treatment with dimethyl sulfoxide (DMSO)" (herein, often abbreviated as "DMSO treatment") means chemical stimulation of dormant eggs by contact treatment with a DMSO solution. , is a method for avoiding the transition to dormancy. The concentration of the DMSO solution used is not limited, but is preferably 90%-100%, 92%-100%, 94%-100%, 96%-100%, or 98%-100%. As used herein, the term "contact" means that all or part of an object is in contact with another object. Therefore, contact with a DMSO solution includes, for example, immersing a dormant egg in a DMSO solution, spraying or applying a DMSO solution to a dormant egg, and the like. The contact time with the DMSO solution is not limited. 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 dormant eggs with water, buffer, or the like in order to remove DMSO.
By this step, diapause is avoided in diapause eggs.

1-3-3. Nucleic Acid Introduction Step The “nucleic acid introduction step” (S0103) is a step of introducing a target nucleic acid into the egg after the dormancy breaking step (S0102). Eggs used in this step are fertilized or unfertilized G0 silkworm eggs.

Many of the eggs into which the nucleic acid is to be introduced are in a state of avoiding dormancy due to the dormancy breaking step (S0102). In this step, it is desirable to introduce nucleic acid into a plurality of eggs after the dormancy breaking step (S0102).

In this step, the "target nucleic acid" is a nucleic acid to be introduced into a silkworm to produce a target transgenic silkworm. For example, DNA and RNA correspond. More specific examples include, but are not limited to, donor DNA/RNA, helper DNA/RNA, or guide DNA/RNA. Preferably, it is a gene of interest, an expression vector containing it, and a nucleic acid that promotes incorporation into the genome, such as a helper plasmid. When introducing a gene, the type of gene does not matter. For example, it may be a gene that encodes a protein or a functional nucleic acid (such as an RNAi molecule). A desired gene that can confer a desired trait can be introduced. At this time, a marker gene may be introduced so that individuals into which the nucleic acid of interest has been introduced can be easily selected in the later-described selection step. In addition, in this step, peptides (including proteins such as enzymes) and/or low-molecular-weight compounds can be introduced along with the nucleic acid, if necessary. In this case, the order of introduction with the nucleic acid does not matter. It may be before nucleic acid introduction, after nucleic acid introduction, or simultaneously with nucleic acid introduction.

The gene to be introduced in this step may be derived from any species. It may be derived from a silkworm, which is the host, or may be a gene derived from an organism of another species, such as a human. As an example, when an antibody is produced using a transgenic silkworm, the foreign DNA can be an expression unit containing an IgG antibody gene and a gene expression regulatory region such as a promoter or terminator.

This step can be performed by a method known in the art for introducing foreign genes into silkworms. For example, if the expression vector is a plasmid with transposon inverted terminal repeats (Handler AM. et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:7520-5) flanking the DNA of interest. For example, the method of Tamura et al. (Tamura T. et al., 2000, Nature Biotechnology, 18, 81-84), the method of Zhou et al. (Zhou W. et al., 2012, Insect Science, 19: 172-182). can be used. Specifically, an injection solution is prepared by dissolving or diluting the expression vector with a solvent such as water or a buffer so that the expression vector has an appropriate concentration. At this time, a helper plasmid having a DNA encoding a transposon transferase is added to the injection solution. Examples of the helper plasmid include pHA3PIG.

Introduction of the nucleic acid of interest into the egg is generally, but not exclusively, carried out using a special pneumatic injection device. For example, 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 according to the type, properties, and purpose of the nucleic acid. Usually 1 nL to 5 nL.

The nucleic acid-introduced eggs may be incubated under suitable conditions, for example, at 25°C until they hatch.

1-3-4. Recombinant Selection Step The “recombinant selection step” (S0104) is a step of selecting genetically modified organisms as genetically modified silkworms from the silkworms generated after the nucleic acid introduction step (S0103).

G0 silkworms that have undergone the nucleic acid introduction step contain genetically modified somatic cells and germ cells, and it is the genetically modified germ cells that are passed on to the next generation. Therefore, this step is performed with G1 silkworms.

When using the G1 fertilized egg obtained from the G0 silkworm of the dormant silkworm line after the nucleic acid introduction step (S0103) through the fertilized egg collection step (S0101), the dormancy breaking step (S0102) is executed for the G1 fertilized egg. and then this step is performed. Silkworms obtained after this step are normal transgenic silkworms. Since gene transfer is not performed in G1 fertilized eggs by microinjection, dormancy breaking treatment for G1 fertilized eggs is not limited to the above-mentioned DMSO treatment, and other known dormancy breaking methods can also be used. For example, pickling treatment, centrifugation treatment, oxygen treatment, and the like can be mentioned.

When using G1 unfertilized eggs obtained through the unfertilized egg collection step (S0105) from the G0 silkworm of the parthenogenetic silkworm line after the nucleic acid introduction step (S0103), this step is for G1 unfertilized eggs. Therefore, the generation induction step (S0106) is performed, and the dormancy breaking step (S0102) is performed. Individuals hatched after the process are cloned silkworms. This step is performed on those hatched cloned silkworms. Therefore, silkworms obtained after this step are transgenic cloned silkworms.

Furthermore, as an exceptional case, even for G0 silkworms that have undergone the fertilized egg collection step, depending on the combination of parent silkworm strains used for the production of G0 silkworms such as crossbreeding, in the G1 egg collection after the nucleic acid introduction step (S0103), A high parthenogenesis rate and non-diapause rate may be expected in G1 eggs collected. In such a case, even if the G0 silkworm has undergone the fertilized egg collection step (S0101), the unfertilized egg collection step (S0105) for collecting unfertilized eggs from the G0 female individual after the nucleic acid introduction step (S0103) is performed. You can also go through This step can also be performed after subjecting the obtained G1 unfertilized egg to the step of inducing parthenogenesis (S0106) and, if necessary, the step of breaking dormancy (S0102). In this case, the silkworm obtained after this step is a genetically modified cloned silkworm.

Selection of genetic recombinants may be performed by methods known in the art. For example, selection may be made based on the trait induced by the nucleic acid of interest introduced in the nucleic acid introduction step (S0103). For example, if the nucleic acid of interest is a foreign gene, prepare genomic DNA or mRNA from the silkworm generated after the nucleic acid introduction step (S0103), and confirm the presence or absence of the foreign gene or the expression of the foreign gene by PCR, etc. do it.

Alternatively, if the expression vector used for nucleic acid transfer contains a marker gene, the desired transgenic 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 new traits not possessed by the host silkworm. Based on the activity of this labeling protein, it becomes possible to easily identify transformants that have the introduced nucleic acid of interest. Here, "based on activity" means based on the result of detection of activity. Detection of the activity may be carried out by directly detecting the activity of the labeled protein itself, or indirectly through metabolites such as dyes generated by the activity of the labeled protein. good. Detection may be by chemical detection (including enzymatic detection), physical detection (including behavioral detection), or sensory detection of the detector (including detection by sight, touch, smell, hearing, taste). Either can be used.

1-3-5. Step of Collecting Unfertilized Eggs The “step of collecting unfertilized eggs” (S0105) is, in principle, a step of collecting unfertilized eggs from individuals of the parthenogenetic silkworm strain. This step is carried out together with the following parthenogenic induction step (S0106) when genetically modified cloned silkworms are produced using a parthenogenic silkworm strain as the parent strain. This step is an essential step for producing transgenic cloned silkworms from parthenogenetic silkworm strains.

In addition, even when the G0 silkworm obtained through the fertilized egg collection step is not a parthenogenic silkworm strain, this step can be performed when G1 eggs obtained from the G0 silkworm can be generated with a high probability by parthenogenesis induction treatment. may also be executed exceptionally.

Therefore, when performing this step and the following parthenogenesis inducing step (S0106), the dormant eggs used in the dormancy breaking step (S0102) are, in principle, dormant unfertilized eggs obtained from individuals of the parthenogenic silkworm strain. As mentioned above, the egg may be, as an exception, a dormant unfertilized egg or a non-dormant unfertilized egg obtained from an individual who has undergone the fertilized egg collection step.

In this step, adult female individuals whose unfertilized eggs have developed into a state capable of parthenogenesis in the body, ie mature unfertilized eggs, are used.

The method for collecting unfertilized eggs is not limited. Examples thereof include a method of collecting by dissection and a method of collecting by natural spawning.

Specifically, for the method of collecting by dissection, for example, after incising the abdomen or tail of a female adult with a scalpel or dissecting scissors, pressure is applied to the abdomen with fingers to remove the ovaries by pushing them out through the incision. good. By immersing the removed ovaries in water, clumped oviducts can be loosened. Subsequently, the ovary is placed on a fine stainless steel mesh or gauze, and rubbed with a finger or the like to separate the fallopian tube tissue and the unfertilized egg. Finally, the oviduct tissue floating on the water is removed by pouring water over the mesh, and the unfertilized eggs remaining at the bottom are collected. By repeating this operation several times, only unfertilized eggs can be recovered.

Unfertilized eggs can also be collected by natural oviposition in which unmated female individuals lay eggs. There is no limitation on the egg collection method. For example, a method is adopted in which an egg-laying mount is given to a female individual after emergence, and eggs are laid on the mount. Specifically, after eclosion, female silkworms are placed at a low temperature of 0 to 10°C, preferably 5°C, and after being stored at that temperature for 1 to 2 days, up to 1 to 7 days, female silkworms are placed at a low temperature. to room temperature (23-28°C), where they are provided with a laying mat and allowed to begin laying under dark conditions.

1-3-6. Parthenogenesis Inducing Step The “parthenogenesis inducing step” (S0106) is a step of subjecting the unfertilized eggs obtained in the unfertilized egg collection step (S0105) to a parthenogenesis induction treatment. This step is a step performed in combination with the unfertilized egg collection step (S0105). Therefore, silkworms obtained through the step of collecting unfertilized eggs (S0105) and this step are clone silkworms.

Parthenogenesis induction treatment may be performed according to a method known in the art, and is not particularly limited. For example, Sugai et al. (Etsuji Sugai et al., 1983, Nihon Seriology Journal, No. 52, No. 1: 51-56), Hirokawa (Masahiko Hirokawa, 1990, Fukushima Sericulture Research Institute, 24: 1-6), and Ozegawa et al. (Kosegawa E., et al. 2012, J Insect Biotechnol Sericology, 81: 37-44) can be referred to for induction methods disclosed. In general, parthenogenesis is induced by applying a physical or chemical stimulus to an unfertilized egg. Specifically, for example, high temperature treatment can be mentioned. As a specific example of the high-temperature treatment, the unfertilized eggs collected in the above step may be exposed to hot water treatment at 45° C. to 50° C. for 15 to 20 minutes. After that, it is preferable to keep the temperature at 12° C. to 18° C. for 2 to 6 days.

The dormant unfertilized egg obtained after this step has already been induced to parthenogenesis. However, they do not hatch because they are dormant eggs. By performing the above-described dormancy breaking step (S0102) on this dormant egg, dormancy is avoided and development continues, so that the egg will eventually hatch.

<Example 1: Production of genetically modified silkworms using dormant eggs>
(the purpose)
The hatching after microinjection and the acquisition of genetically modified silkworms by the method for producing genetically modified silkworms of the present invention are verified.

(Method)
In each experimental plot, the dormant silkworm strain ``Ariake'' was assigned to two experimental plots (experimental plots A and B), the dimorphic strain ``Hi 137'' was assigned to one experimental plot (experimental plot C), The sex line ``branch 146'' was used for one experimental group (experimental group D). These strains were obtained from the National Agriculture and Food Research Organization of Japan.

In each experimental plot, the male and female moths of the above strains were mated in the same strain, and then reared at 5°C for 1 to 3 days. At the time of egg collection, female moths were transferred to an egg-laying mat and allowed to lay eggs. All the eggs obtained here are dormant eggs. After egg collection, DMSO treatment was performed.

The DMSO treatment was performed by putting the eggs collected for each experimental group into a petri dish and immersing them in 100% DMSO (Fuji Film Wako Pure Chemical Industries, Ltd.). 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. The treated eggs were washed with water and then air dried.

Subsequently, nucleic acid was introduced into DMSO-treated eggs by microinjection. Nucleic acids for experimental groups A, C and D were 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), and in experimental group B, pHA3PIG and the vector plasmid pBac [3xP3-EGFPafm] (Horn and Wimmer, 2000) were treated with DMSO 4-6 hours after spawning. introduced into eggs. Post-injection eggs were incubated in a humidified plastic box at 25°C until hatching. The ratio of hatched eggs to the number of injected eggs was calculated as the hatching rate.

Larvae after hatching in each experimental group were transferred to a plastic box containing artificial feed and reared at 25 to 27°C until they became adults. Adults of G0 (0th generation after injection) obtained for each experimental group were mated with each other to obtain G1 (1st generation after injection) eggs. At this time, one moth colony was defined as an aggregate of about 300 eggs obtained from one female. G1 eggs were immersed in 6N HCl for 1 hour to break dormancy, washed with water, and then incubated in a moist box at 25°C to promote development. When each egg was irradiated with excitation light and fluorescence of a marker protein (DsRed or GFP) based on the marker gene introduced monocularly was detected, the fluorescent egg was transferred to a Petri dish. Larvae hatched from the transferred eggs were bred to establish transgenic silkworms. A moth plot in which one or more genetically modified silkworms were obtained was defined as a recombinant moth plot, and the ratio of the recombinant moth plot to the moth plot was calculated as the recombinant silkworm acquisition rate (recombination rate).

(result)
Table 1 shows the above results.

From the above results, if dormancy was broken by DMSO treatment, a hatching rate of 14.4 to 62.7% was obtained even after microinjection, regardless of the strain, type of introduced gene, and DMSO treatment time. Zhao AC et al. (Non-Patent Document 3), when microinjected into eggs that had undergone dormancy breaking by acid pickling, the subsequent hatching rate was only 3.4 to 4.6%. It was an unexpected result that a hatching rate of 3 to 10 times higher was obtained. In addition, as a result of confirming the emergence of genetically modified silkworms in hatched individuals in each moth plot, transgenic silkworms were obtained in approximately 20-40% of moth plots in all experimental plots.

Claims (6)

A method for producing a genetically modified silkworm, comprising:
A fertilized egg collection step of collecting fertilized eggs from individuals of the dormant silkworm strain,
A dormancy breaking step of breaking dormancy by contacting the fertilized eggs collected in the fertilized egg collecting step with dimethyl sulfoxide,
A nucleic acid introduction step of introducing a target nucleic acid into the egg after the dormancy breaking step by a microinjection method, and a recombinant selection step of selecting a genetically modified organism from next-generation silkworms after the nucleic acid introduction step. How to make. A method for producing a genetically modified silkworm, comprising:
An unfertilized egg collection step of collecting unfertilized eggs from individuals of the parthenogenetic silkworm strain,
a parthenogenesis induction step of subjecting the unfertilized egg collected in the unfertilized egg collection step to a parthenogenesis induction treatment;
a dormancy-breaking step of breaking dormancy by contacting the unfertilized egg after the parthenogenesis-inducing step with dimethylsulfoxide;
A nucleic acid introduction step of introducing a target nucleic acid into the egg after the dormancy breaking step by a microinjection method, and a recombinant selection step of selecting a genetically modified organism from next-generation silkworms after the nucleic acid introduction step. How to make. 3. The production method according to claim 1, wherein the contact treatment time with the dimethylsulfoxide is 10 minutes to 1 hour. The production method according to any one of claims 1 to 3, wherein a labeling gene is further introduced in the nucleic acid introduction step. 5. The production method according to claim 4, wherein the selection in the recombinant selection step is based on the expression of the marker gene. The production method according to any one of claims 2 to 5, wherein the parthenogenetic induction treatment is a high-temperature treatment in which unfertilized eggs are exposed to 45°C to 50°C for 15 to 20 minutes.

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