Temporary treatment medium, treatment kit, agent for inhibiting embryonic development arrest, method for inhibiting embryonic development arrest, method for producing developmentally engineered products, transplantation method, and treatment method

Abstract

Provided is a temporary treatment medium for inhibiting embryo developmental arrest due to manipulation of an embryo or the like. A temporary treatment medium according to the present invention reduces damage to an in-vitro culture due to manipulation thereof, said in-vitro culture containing one of pluripotent stem cells, reproductive cells, a fertilized egg, and an embryo or any combination thereof. For this purpose, the temporary treatment medium contains a cytoskeleton regulator and / or an apoptosis inhibitor. The cytoskeleton regulator and / or the apoptosis inhibitor is preferably an Rho kinase inhibitor. Specifically, a Rock inhibitor can be used as the Rho kinase inhibitor. The Rock inhibitor is, for example, Y-27632. The temporary treatment medium is used for treating an in-vitro culture for a specific period before and / or after manipulation involving damage thereto.

Description

[Technical Field]

[0001] In particular, the present invention relates to a temporary treatment medium for performing temporary treatment on in vitro cultures, a treatment kit, an agent for suppressing embryonic development arrest, a method for suppressing embryonic development arrest, a method for producing a developmentally engineered product, a transplantation method, a treatment method, and a developmentally engineered product. [Background technology]

[0002] Traditionally, developmental engineering and reproductive medicine have been widely used in humans, mice, rats, rabbits, pigs, cattle, horses, monkeys, and other animals. While gamete freezing and thawing techniques are essential, they can be inefficient in some species. When implementing reproductive technologies, there is a need for techniques to stabilize gametes, such as eggs, and enable them to withstand freezing and thawing procedures, ICSI, nuclear transfer, chimeric embryo production, and gene transfer procedures. For example, in recent years, there have been many reports of the creation of genetically modified animals using genome editing techniques such as CRISPR-Cas, as part of fundamental research for the creation of disease model animals and gene therapy models. In order to produce genetically modified animals, it is necessary to culture embryos in vitro and manipulate them, such as by modifying their genes. However, because embryos cultured and manipulated outside the body are subject to damage, it is often not possible to produce individual animals, depending on the strain or species.

[0003] Previously, Non-Patent Document 1 has described that enhancing the activity of Rho kinase by, for example, introducing recombinant Rho kinase into cells can increase the survival rate of embryos whose cytoskeleton has been damaged by freezing. Rho kinase is a type of serine-threonine protein kinase and has two highly homologous isoforms, ROCK1 and ROCK2. Both act on the cytoskeleton and are involved in important physiological functions, including regulating cell shape and movement. According to Non-Patent Document 1, the use of a "Rho kinase inhibitor" that suppresses the action of Rho kinase significantly reduces the survival rate of embryos (see the abstract of Non-Patent Document 1, etc.).

[0004] Meanwhile, Patent Document 1 describes a method for forming retinal pigment epithelial cells from pluripotent stem cells such as iPS cells and ES cells using Y27632, a type of Rho kinase inhibitor. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 164240 [Patent Document 2] Japanese Patent Application Laid-Open No. 2015-70825 [Non-patent literature]

[0006] [Non-Patent Document 1] Gu et al., “Rho / RhoA-associated kinase pathway improves the anti-freezing potential of murine hatched and diapaused blastocysts,” Sci Rep, (US), 2017, 7:6705 [Non-patent document 2] Horii, T et al., “Efficient generation of conditional knockout mice via sequential introduction of lox sites,” Sci Rep, (US), 7, :7891. Summary of the Invention [Problem to be solved by the invention]

[0007] However, the enhancement of Rho kinase described in Non-Patent Document 1 was unable to sufficiently suppress embryonic damage during the production of genetically modified animals, making it extremely difficult to produce individuals from strains that are sensitive to artificial embryo manipulations that involve damage.

[0008] The present invention has been made in view of the above circumstances, and an object of the present invention is to solve the above-mentioned problems. [Means for solving the problem]

[0009] The temporary treatment medium of the present invention comprises: A temporary treatment medium for reducing damage caused by manipulation to an in vitro culture containing any one or any combination of germ cells, fertilized eggs, and embryos excluding primordial germ cells, the medium containing a cytoskeleton regulator and / or an apoptosis inhibitor, the cytoskeleton regulator and / or the apoptosis inhibitor being a Rho kinase inhibitor, the manipulation being a treatment that causes damage to the in vitro culture, excluding freezing and thawing, the damage being damage or alteration of various structures necessary for cell survival and normal differentiation, and causing double-strand breaks in DNA in the nucleus. It is characterized by: The treatment kit of the present invention is characterized by containing the temporary treatment medium. The embryonic development arrest inhibitor of the present invention comprises: The method comprises containing an apoptosis inhibitor, reducing damage caused by manipulation to an in vitro culture and suppressing embryonic development arrest, the in vitro culture comprising any one or any combination of germ cells excluding primordial germ cells, fertilized eggs, and embryos, the cytoskeleton regulator and / or the apoptosis inhibitor is a Rho kinase inhibitor, the manipulation is a treatment that causes damage to the in vitro culture, excluding freezing and thawing, and the damage is accompanied by damage or alteration of various structures necessary for cell survival and normal differentiation, and double-strand breaks in DNA in the nucleus. It is characterized by: The method for suppressing embryonic development arrest of the present invention comprises: A method for suppressing embryonic development arrest by reducing damage caused by manipulation to an in vitro culture containing any one or any combination of germ cells excluding primordial germ cells, fertilized eggs, and embryos, and suppressing embryonic development arrest, the method comprising treating the in vitro culture with a temporary treatment medium containing a cytoskeleton regulator and / or an apoptosis inhibitor for a specific period before and / or after the manipulation that causes damage to the in vitro culture after it has been obtained outside the body, the cytoskeleton regulator and / or the apoptosis inhibitor is a Rho kinase inhibitor, the manipulation is a treatment that causes damage to the in vitro culture, excluding freezing and thawing, the damage involves damage or alteration of various structures necessary for cell survival and normal differentiation, and double-strand breaks in DNA in the nucleus, and the human fertilized eggs and embryos are those that are less than 14 days old from fertilization and will not develop into individuals. It is characterized by: The method for producing a developmentally engineered product of the present invention comprises the steps of: Excluding humans The method is characterized by producing a developmentally engineered product including any one or any combination of an individual, an organ, a tissue, and a cell. The transplantation method of the present invention is a method for transplanting the in vitro culture treated by the method for suppressing embryonic development arrest and / or the developmentally engineered product produced by the method for producing a developmentally engineered product. , to recipients other than humans It is characterized by transplantation. The treatment method of the present invention comprises: Excluding humans A method for treating a mammal, comprising: treating the in vitro culture treated by the method for suppressing embryonic development arrest and / or the developmentally engineered product produced by the method for producing a developmentally engineered product. , to recipients other than humans It is characterized by transplantation. [Effects of the Invention]

[0010] According to the present invention, a treatment medium can be provided that enables the production of individuals even from strains that are sensitive to artificial embryo manipulations that involve damage. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a conceptual diagram showing the flow of a method for suppressing embryonic development arrest according to an embodiment of the present invention. [Figure 2]1 is a table summarizing the lines and results of operations that made it possible to produce individuals using the method for suppressing embryonic development arrest and the method for producing developmentally engineered products according to Example 1 of the present invention. [Figure 3] 1 is a table showing an example of producing knockout mice by electroporation using C57BL / 6J mice according to Example 1 of the present invention. [Figure 4] 1 shows electrophoresis photographs confirming Flox mice using the 2STEP method using C57BL / 6J according to Example 1 of the present invention. [Figure 5] 1 is a photograph of an individual mouse developed from a frozen egg of the inbred strain SPR2 (inbred strain SPR2 heterogeneous mouse) of xenogeneic mouse Mus spretus (Algerian house mouse) according to Example 1 of the present invention. [Figure 6] 1 shows a photograph of ES cells established in a xenogeneic mouse, Mus caroli, according to Example 1 of the present invention. [Figure 7] 1 is a photograph of electrophoresis confirming Tyr gene knockout Mus caroli according to Example 1 of the present invention. [Figure 8A] 1 is a table showing the state of embryos obtained by genome editing of inbred rats according to Example 1 of the present invention. [Figure 8B] 1 is a table showing the state of embryos obtained by genome editing of inbred rats according to Example 1 of the present invention. [Figure 8C] 1 is a table showing the state of embryos obtained by genome editing of inbred rats according to Example 1 of the present invention. [Figure 9A] 1 is a photograph of an individual generated by genome editing of the heterologous inbred mouse strain SPR2 according to Example 1 of the present invention. [Figure 9B] 1 is a photograph of an individual generated by genome editing of the heterologous inbred mouse strain SPR2 according to Example 1 of the present invention. [Figure 9C] 1 is a photograph of an individual generated by genome editing of the heterologous inbred mouse strain SPR2 according to Example 1 of the present invention. [Figure 10] 1 shows photographs of individuals generated by performing homozygous large deletion knockout on an inbred rat strain by electroporation according to Example 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0012] <Embodiment> To produce genetically modified animals, etc., it is necessary to culture pluripotent stem cells, germ cells, fertilized eggs, embryos, etc. (hereinafter referred to as "in vitro cultures") outside the body and then treat, process, manipulate, etc. (hereinafter simply referred to as "manipulation") using various techniques, such as molecular biology, genetic engineering, reproductive engineering, developmental engineering, reproductive medicine, regenerative medicine, and other technologies. However, manipulation of in vitro cultures can damage the cells, causing the developmental stage to stop (hereinafter referred to as "embryonic development arrest") depending on the strain or animal species. For this reason, it has often been impossible to produce an individual animal. Therefore, the inventors conducted extensive experiments and discovered that by treating cells damaged by manipulation with a temporary treatment medium containing an intracellular skeleton regulator and / or an apoptosis inhibitor, it is possible to reduce the damage and suppress the arrest of embryonic development, thereby completing the present invention.

[0013] [Primary treatment medium] The temporary treatment medium of this embodiment is a temporary treatment medium for reducing damage caused by manipulation to in vitro cultures, and is characterized by containing a cytoskeleton regulator and / or an apoptosis inhibitor. Here, the in vitro culture of this embodiment includes any one or any combination of animal pluripotent stem cells, germ cells, fertilized eggs, and embryos used in molecular biology, genetic engineering, reproductive engineering, developmental engineering, reproductive medicine, regenerative medicine, etc. (hereinafter referred to as "developmental engineering, etc.") and used for culture and various operations outside the body.

[0014] Pluripotent stem cells of this embodiment include stem cells with pluripotency that can differentiate into various cell types in organisms such as mammals, including humans, and other vertebrates. Here, the pluripotent stem cells of this embodiment are preferably passageable, maintain a state in which differentiation does not progress even after passage, and are resistant to changes in karyotype, etc., or epigenetic phenotype. In this regard, the pluripotent stem cells of this embodiment are preferably resistant to changes in proliferation capacity in vitro or in vivo. Specific examples of such pluripotent stem cells of this embodiment include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and other artificially generated or selected stem cells with pluripotency. These pluripotent stem cells of this embodiment may also be stem cells created by reprogramming somatic cells using various vectors, such as retroviruses, adenoviruses, and plasmids, containing specific genes, RNA, low-molecular-weight compounds, etc.

[0015] In addition, the pluripotent stem cells of this embodiment do not necessarily have to be cells with pluripotency close to totipotency, but naive cells with higher pluripotency than usual can also be used. Furthermore, the pluripotent stem cells of this embodiment preferably have the ability to differentiate into developmentally engineered products described below, such as by developing into an individual by chimerizing with a germ cell described below. Furthermore, the pluripotent stem cells of this embodiment can be cultured and maintained on feeder cells or on cell culture plates coated with a basement membrane matrix such as collagen, and then harvested or frozen and stored.

[0016] Furthermore, the pluripotent stem cells of this embodiment may be cells created from cells obtained from a patient with a disease, cells that serve as models for other diseases, cells into which a reporter gene has been incorporated (reporter cells), cells capable of conditional knockout or knock-in, other genetically modified cells, etc. This genetic modification includes the addition, modification, or deletion of genes within chromosomes, the addition of genes using various vectors or artificial chromosomes, changes in epigenetic regulation, the addition of artificial genetic material such as PNA, and other genetic modifications.

[0017] The germ cells of this embodiment include primordial germ cells, spermatogonia, oogonia, germ cells before meiosis, other cells derived from germ cells, egg cells, sperm, cells capable of parthenogenesis, cells capable of teratoma formation different from the above-mentioned pluripotent stem cells, and other cells that can undergo ontogeny through some manipulation. The germ cells of this embodiment may also be cryopreserved and then thawed.

[0018] The fertilized egg in this embodiment is a fertilized egg formed by fertilizing an animal egg cell with a sperm, a parthenogenetic egg, or other egg-like cell capable of development. This fertilized egg may be one fertilized by in vitro fertilization or intracytoplasmic sperm injection, or may be a cell obtained at the time when cleavage has begun and the egg is fully competent (a cell from a cloned fertilized egg), or may be a frozen and thawed fertilized egg. In this embodiment, other types of fertilized eggs commonly used by those skilled in the art may also be used.

[0019] The embryo of this embodiment is a cell mass at a stage where the number of cells has increased from a fertilized egg by cleavage and reached a certain number. The embryo of this embodiment can also be a cleaved embryo, for example, an embryo obtained by bisecting a two-cell stage embryo. Alternatively, the embryo of this embodiment may be a cleaved embryo, for example, an embryo obtained by bisecting a two-cell stage embryo, or may be one that has developed into a morula or gastrula. Alternatively, the embryo of this embodiment may be a blastocyst, etc. Here, the embryo of this embodiment may be an embryo derived from a totipotent cell created using primordial germ cells or the like created from pluripotent stem cells. In addition, the mammalian embryo that is the subject of this embodiment may be a chimera in which cells from multiple types of animals are mixed. In this case, the chimeric embryo may also be combined with cells from an animal other than a mammal. Furthermore, the subject of embryo processing in this embodiment is not limited to simple embryos, but can also be applied to embryoid bodies that do not necessarily develop into individuals or fetuses but differentiate only into tissues and organs of each lineage. In this case, embryos from which a portion has been removed for analysis, i.e., biopsied embryos, can also be applied. Furthermore, the embryos used in this embodiment may be cryopreserved and then thawed. The cryopreserved embryos may be blastocysts, which are common to those skilled in the art. That is, the in vitro cultures according to this embodiment may be cryopreserved and then thawed.

[0020] The method for obtaining the embryo according to this embodiment is not particularly limited. For example, the embryo according to this embodiment can be obtained by in vivo fertilization, in vitro fertilization, or nuclear transfer. Additionally, the mammalian embryo of this embodiment may be a mammalian embryo whose genetic information has been processed by techniques such as transgenics, gene knockout, conditional knockout, etc., in which a gene has been introduced by genetic recombination techniques using various vectors, etc. This processing of genetic information may involve gene introduction or removal into the genome by genome editing, etc., extrachromosomal gene introduction using a plasmid or artificial chromosome, epigenetic control such as control of methylation of specific chromosomal sites or histone modification, addition of PNA or artificial bases, or various other genetic information processing techniques may be used.

[0021] Here, the in vitro culture of this embodiment may be one selected in the form of a colony or the like using various markers or visual inspection. The in vitro culture of this embodiment may also be a mixed cell population, tissue, organ, or the like (hereinafter referred to as "tissue, etc."). These in vitro cultures may contain a mixture of cells in various states of differentiation and development. That is, each cell belonging to the in vitro culture may be in a developmental stage and not fully differentiated or may be immature.

[0022] The animals to which the in vitro culture of this embodiment belongs are not particularly limited and broadly include vertebrates and invertebrates, including fish, amphibians, reptiles, birds, and mammals. Mammals that are the subject of embryo treatment in this embodiment are, for example, from the order Primates, rodents, lagomorpha, cetaceans, perissodactyla, or carnivora, and can be treated with a temporary treatment medium of this embodiment prepared for embryos of different orders or species. Note that the present embodiment can also be applied to embryos of placental mammals of the subclass Eutheria, including rare mammals outside these orders, as subjects of embryo treatment.

[0023] The animals to which the in vitro culture of this embodiment belongs can be described from a different perspective than the above-mentioned order units, and examples thereof include humans (Homo sapiens), laboratory animals, livestock, and companion animals. Among these, laboratory animals include rodents such as mice (Mus musculus), rats (Rattus norvegicus), hamsters (Mesocricetus auratus), and guinea pigs (Cavia porcellus). Examples of lagomorphs include rabbits (Leporinae Trouessart). Examples of livestock include cetaceans such as pigs (Sus scrofa domesticus), cattle (Bos taurus), and sheep (Ovis aries). Examples of odd-toed ungulates include horses (Equus caballus). Furthermore, examples of companion animals include animals of the order Carnivora, such as cats (Felis silvestris catus), dogs (Canis lupus familiaris), and ferrets (Mustela putorius). Examples of non-human primates include apes such as gorillas (Gorilla), chimpanzees (Pan troglodytes), and rhesus monkeys (Macaca mulatta), as well as other primates. Other examples include animals of different species from the above examples, such as rodents, such as the Okinawan house mouse (Mus caroli) and the Algerian house mouse (Mus spretus), as well as their subspecies, as described in the Examples below. Among these, the subspecies of this embodiment may be taxonomic subspecies, or ones whose mitochondrial DNA sequences, etc., differ at least partially from existing ones. Furthermore, species or subspecies with variations in genomic information may be designated as so-called "wild species." In addition, different species of animals from the other orders mentioned above may also be included. In this embodiment, for convenience, these different species of animals are referred to in correspondence with animals that are similar in appearance and characteristics, such as "different species of mice," "different species of rats," and "different species of cats." Furthermore, the above-mentioned classifications of laboratory animals, livestock, companion animals, etc. are for convenience only, and each is used for different purposes, such as breeding and medical purposes.Furthermore, the animals according to this embodiment may be animals that have already been genetically modified, animals that have been genetically modified through breeding, pedigreed animals, or other genetically modified animals. Furthermore, the animals of this embodiment include a wide range of invertebrates and animals that develop through cleavage, such as chordates, mollusks, annelids, and arthropods.

[0024] The animal to which the in vitro culture of this embodiment belongs may be derived from a mammalian lineage that is particularly sensitive to the procedures of this embodiment and prone to embryonic arrest. Furthermore, as shown in the examples below, such mammals may be hybrids, which are crossbreeds with a clear genetic background, or inbred strains, which are strains of animals and plants obtained by inbreeding primarily between siblings or older siblings for 20 or more generations. Furthermore, they may be mutant (genetically mutated) animals such as nude mice, specific disease model animals, heterologous animals that have been altered as species, hybrid animals between species, etc. Furthermore, they also include animals and subspecies that have been artificially bred or fixed as species.

[0025] Here, the manipulation of in vitro cultures in this embodiment (such as artificial embryo manipulation, hereinafter simply referred to as "manipulation") includes treatments that damage in vitro cultures, which are necessary for developmental engineering and the like. This manipulation includes not only manipulation of the in vitro cultures themselves, but also manipulation of the mother and germ cells associated with obtaining the in vitro cultures. Damage to in vitro cultures also includes damage associated with various manipulations, such as experiments and treatments, performed after obtaining the in vitro cultures. Specifically, this damage includes damage or alteration to various structures necessary for cell survival and normal differentiation, such as intracellular organelles including the nucleus, DNA including genes, the cytoskeleton, various other intracellular structures, the cell membrane, and the extracellular matrix, which is an extracellular structure. In particular, in this embodiment, manipulations may involve physical or organic stress on the cells, ionization, damage by reactive oxygen species or chemicals associated with destruction of intracellular organelles, etc., resulting in nicks or double strand breaks (DSBs) in intranuclear DNA. These damages may be at least partially recoverable by cell metabolism. Conversely, if cells do not recover from damage, they may undergo cell cycle arrest, differentiation arrest or change, apoptosis, necrosis, or other abnormal states.

[0026] Specifically, the manipulation of the in vitro culture of this embodiment includes, for example, treatments that damage embryos (cells), such as superovulation treatment of the mother to increase embryo availability, freezing or thawing of embryos associated with transplantation of frozen embryos, cell dissociation, nuclear transplantation (NT), intracytoplasmic sperm injection (ICSI), microinjection (MI), and electroporation (EP). Furthermore, treatments that damage the nucleus and DNA within the nucleus include exposure to large amounts or high molecular weight (long sequences) DNA or RNA, and treatments that involve multiple DSBs, as described below. Furthermore, manipulations such as cell fusion are also included in the manipulation of this embodiment. Cell fusion, etc., also includes the creation of polyploid cells, such as aneuploids and tetraploids.

[0027] In addition, the manipulation of the in vitro culture of this embodiment includes various treatments such as osmotic pressure changes, perforation with other chemicals, perforation with forceps, and in vitro fertilization (IVF). As described above, these treatments include those performed to introduce nuclei, chromosomes, DNA, RNA, and the like into the in vitro culture for purposes such as developmental engineering. This introduction may be performed using various vehicles. Examples of such vehicles include plasmids, viral vectors, liposomes, and other drug delivery systems (DDSs), as well as various techniques for introducing other macromolecules into cells. Among these, viral vectors may be constructed using viruses commonly known to those skilled in the art, such as adenovirus, adeno-associated virus, and retrovirus. Furthermore, the above-described manipulations may be performed when using these vehicles. Alternatively, the above-described manipulations may be performed to produce the above-described pluripotent stem cells, etc. Note that various treatments related to in vitro maturation of immature oocytes (IVM), blastocyst transfer (BT), and the like may also be included in the manipulations of this embodiment. This embryo transfer includes surgical transfer into the fallopian tube, non-surgical transfer into the uterus, and the like, which are well known to those skilled in the art.

[0028] More specifically, the manipulation of the in vitro culture in this embodiment includes various techniques that cause significant damage to the in vitro culture, such as treatments involving multiple DSBs. Examples of such treatments involving multiple DSBs include the creation of animals with multiple gene mutations. These multiple gene mutations also include techniques for creating gene knockout, knockin, and conditional knockout animals. More specifically, the method includes the 2STEP method for creating conditional knockout animals. The 2STEP method involves sequential electroporation to insert Flox sequences into two locations by genome editing (see, for example, Non-Patent Document 2). This method allows the creation of animals (Flox animals) with a locus in which the target gene region is flanked by Cre recombinase target sequences, loxP.

[0029] The cytoskeleton regulator of this embodiment is a substance that regulates the polymerization, depolymerization, assembly, dissociation, and behavior of proteins associated with the cytoskeleton. The cytoskeleton of this embodiment includes polymers and aggregates of structural proteins, such as actin filaments, microtubules, and intermediate filaments, which constitute the cytoskeleton, nuclear skeleton, membrane structures, and other intracellular skeletal structures. Specifically, the cytoskeleton regulator of this embodiment may be, for example, a substance involved in the polymerization and depolymerization of actin and myosin inside and outside the nucleus, which are involved in DSB repair. It is known that DSB repair activity varies depending on the cell cycle, and that the major cell cycles from fertilized eggs to early embryos differ from those of somatic cells during development. Therefore, the cytoskeleton regulator of this embodiment can be a substance that regulates the cell cycle in accordance with the duration and proportion of these cell cycles. In other words, by regulating the polymerization, depolymerization, assembly, dissociation, and other functions of the cytoskeleton using the cytoskeleton regulator, it is possible to promote DSB repair of DNA in the nucleus damaged by the procedures described below. This makes it possible to suppress developmental arrest due to damage.

[0030] Furthermore, the apoptosis inhibitor of this embodiment is a substance that delays and / or suppresses apoptosis of cells in the developmental or maintenance stage. This includes substances that have the effect or action of delaying and / or suppressing apoptosis in association with DSB repair or cell cycle regulation by the above-mentioned cytoskeleton regulator. That is, the cytoskeleton regulator and the apoptosis inhibitor may be the same substance.

[0031] Here, the cytoskeleton regulator and / or apoptosis inhibitor of this embodiment is, for example, a protein, a nucleic acid, a low molecular weight compound, or any other organic compound, and is not particularly limited.

[0032] In this embodiment, it is preferable to use, for example, an inhibitor of Rho kinase (Rock1 or Rock2) as the cytoskeleton regulator and / or apoptosis inhibitor. As the Rho kinase inhibitor, for example, a Rock inhibitor is preferably used. Examples of the ROCK inhibitor include, but are not limited to, Y-27632 (trans-4-[(1R)-1-Aminoethyl]-N-4-pyridinylcyclohexanecarboxamide), Fasudil (1-(5-isoquinolinesulfonyl)homopiperazine), and H-1152 ((S)-(+)-4-Glycyl-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine). Other ROCK inhibitors can also be suitably used.

[0033] The concentration of the Rock inhibitor contained in the temporary treatment medium of this embodiment can be appropriately determined by those skilled in the art depending on the type, state, and density of the in vitro culture, the type and content of the operation, and other conditions. This Rock inhibitor concentration may be as low as about 1 / 2 to 1 / 100 of the "optimal" concentration of 20 μM shown in Non-Patent Document 1 (see, for example, p. 6, paragraph 4 of Non-Patent Document 1). In other words, the Rock inhibitor does not necessarily have to be present at a concentration that completely inhibits Rho kinase activity. Specifically, the concentration of the Rock inhibitor contained in the temporary treatment medium of this embodiment is, for example, 0.1 μM to 20 μM, preferably 5 μM to 15 μM, and more preferably 8 μM to 12 μM, for example, Y-27632. In the Examples described below, an example is described in which temporary treatment is performed using a temporary treatment medium containing Y-27632 at a concentration of 10 μM.

[0034] In addition, in this embodiment, it is also possible to use an cytoskeleton regulator that has no or little apoptosis-inhibitory effect in normal cells. In this embodiment, for example, cytochalasin B (CAS number: 14930-96-2, 2H-Oxacyclotetradecino[2,3-d]isoindole-2,18(5H)-dione, 6,7,8,9,10,12a,13,14,15,15a,16,17-dodecahydro-5,13-dihydroxy-9,15-dimethyl-14-methylene-16-(phenylmethyl)-, (3E,5R,9R,11E,12aS,13S,15S,15aS,16S,18aS)-) can be used as such an cytoskeleton regulator. When cytochalasin B is used, a similar effect can be obtained even at a dose of 1 / 20 to 1 / 5 of that of Y-27632. Specifically, the cytochalasin B is preferably 0.01 μM to 15 μM, preferably 1 μM to 12 μM, and more preferably 3 μM to 8 μM. In other words, better results can be obtained with an amount of cytochalasin B several μM less than that of Y-27632.

[0035] In the case of other cytoskeleton regulators and / or apoptosis inhibitors, the concentration can be set within the above-mentioned ranges. In addition, the concentration of the cytoskeleton regulator and / or apoptosis inhibitor of this embodiment may be constant during each period described below, may be changed during each period, or may be changed stepwise during each period.

[0036] Additionally, the cytoskeleton modulating agent and / or apoptosis inhibitor according to the present embodiment is an example of an embryonic development arrest inhibitor according to the present embodiment. As described above, these embryonic development arrest inhibitors reduce damage caused by manipulation to in vitro cultures containing any one or any combination of pluripotent stem cells, germ cells, fertilized eggs, and embryos, and suppress embryonic development arrest. Furthermore, the embryonic development arrest inhibitor according to the present embodiment is used for the temporary treatment medium according to the present embodiment.

[0037] In addition, the temporary treatment medium of this embodiment contains components according to the type, state, density, type and content of manipulation of the in vitro culture, other conditions, and the like. The components may include, for example, components necessary for constituting a medium for culturing an in vitro culture and water. For example, the primary treatment medium of this embodiment may be used by adding pH buffer compounds, amino acids, vitamins, antioxidants, antibiotics, collagen precursors, trace metal ions or complexes, various salts, etc. More specifically, the temporary treatment medium of this embodiment may contain, for example, components of a medium commonly used by those skilled in the art for in vitro culture. Examples of such a medium include a general medium such as DMEM (Dulbecco's Modified Eagle Medium). Alternatively, a medium containing specific components specialized for pluripotent stem cells, germ cells, fertilized eggs, embryos, etc. may be used. Additionally, the medium may contain serum or various serum substitutes. These media containing various serum substitutes may be used in xeno-free (Xeno-Free, XF, or Animal Component-Free, ACF) culture systems.

[0038] Furthermore, the medium may also contain various RNAs, peptides, proteins, etc. for promoting differentiation and growth. These include various differentiation-inducing factors, growth factors, etc. Furthermore, depending on the type of manipulation, the medium may also contain low molecular weight compounds for differentiation induction, such as retinoic acid, as required. Alternatively, the temporary treatment medium of this embodiment may contain only components that prevent cells from dying in a short period of time, such as PBS.

[0039] [Processing kit] The processing kit of this embodiment is characterized by comprising the above-mentioned temporary processing medium. In addition, the processing kit of this embodiment may also contain operating solutions (liquids) for various operations, normal culture media used during normal culture, operating media for different types of operations (hereinafter referred to as "operating solutions"), and other reagents required for operations. Such reagents include, for example, the probes and primers of this embodiment, various enzymes, buffer solutions, washing solutions, lysis solutions, and testing reagents. Additionally, in vitro cultures, containers, other materials, equipment, tools, etc. required for the operation of this embodiment may be added and provided as a processing kit of this embodiment. Furthermore, the processing kit of this embodiment may also include reagents, food, cages, drinking water, and other items for maintaining the developmentally engineered product described below. In addition, it is also possible to provide a treatment kit configured to contain carriers and other reagents necessary for the treatment described below. Furthermore, it is also possible to provide a treatment kit that includes an intracellular skeleton regulator and / or an apoptosis inhibitor that can be added to a normal culture medium to form a temporary treatment medium, along with a manual describing the concentration and treatment method.

[0040] [Method for inhibiting embryonic development arrest] The method for suppressing embryonic development arrest of this embodiment is a method for suppressing embryonic development arrest by reducing damage caused by manipulation of in vitro cultures. Specifically, the method for suppressing embryonic development arrest of this embodiment is characterized by treating the in vitro cultures with a temporary treatment medium containing a cytoskeleton regulator and / or an apoptosis inhibitor for a specific period before and / or after manipulation.

[0041] Specifically, the main flow of the method for suppressing embryonic development arrest of this embodiment will be described with reference to FIG. In the method for inhibiting embryonic development arrest of this embodiment, it is preferable to appropriately set the duration and period of treatment. Specifically, after preparing an in vitro culture, temporary treatment is performed in the temporary treatment medium of this embodiment for a first specific period. Thereafter, the medium is changed to a normal medium or an operating solution for a first waiting period, and operation is performed after waiting. Thereafter, the medium is changed to the normal medium of this embodiment for a second specific period, and operation is performed after waiting. Then, the medium is changed to the temporary treatment medium of this embodiment, and temporary treatment is performed again. Furthermore, in some cases, a third waiting period is waited before the subsequent treatment.

[0042] More specifically, in the method for suppressing embryonic development arrest of this embodiment, an in vitro culture to be treated is first prepared. This preparation includes various preparatory steps such as collection of the in vitro culture, thawing of a frozen culture, picking up colonies, dissociating cell aggregates, and preparing drops by covering the culture solution with mineral oil or the like.

[0043] Next, the prepared in vitro culture is treated with the temporary treatment medium of this embodiment for a first specific period prior to manipulation. This first specific period can be appropriately set depending on the type of in vitro culture and the type of manipulation to be performed later. The first specific period is, for example, preferably about 1 minute to 2 hours, more preferably about 15 minutes to 1 hour, and even more preferably about 30 minutes to 1 hour. By performing this temporary treatment for the first specific period, the effect of suppressing embryonic development arrest in the in vitro culture can be enhanced. Note that, depending on the species or strain, the temporary treatment for the first specific period may not be necessary.

[0044] Next, the in vitro culture treated with this temporary treatment medium is recovered using a centrifuge or the like, washed with a washing solution such as minimal medium containing little serum or PBS (Phosphate Buffered Saline), and then recovered again and transferred to a normal medium. This normal medium is a medium commonly used prior to manipulation, and is appropriately selected depending on the type of manipulation, such as DMEM medium, serum-free medium, or PBS itself. The in vitro culture transferred to the normal medium is then left to stand for a first waiting period before manipulation. This first waiting period is preferably, for example, about 1 minute to 2 hours, and more preferably about 15 minutes to 1 hour. If it is longer than this range, the effect of inhibiting embryonic development arrest in the in vitro culture may not be sufficiently achieved. This first waiting period may be omitted. That is, the temporary treatment medium may be washed, replaced with an operating solution (such as an operating medium) or a normal medium, and the in vitro culture product may be added, followed by the operation.

[0045] Next, the in vitro culture is subjected to the above-mentioned manipulation. This manipulation may cause damage to the in vitro culture. Depending on the type of manipulation, the in vitro culture may be placed in a manipulation solution before the manipulation. Depending on the type of manipulation, the manipulation may be performed in normal medium or temporary treatment medium.

[0046] Thereafter, the manipulated in vitro culture is recovered, transferred to a standard medium, and left to stand for a second waiting period. This second waiting period can be set appropriately depending on the type and content of the manipulation. Specifically, this second waiting period is preferably, for example, about 1 minute to 2 hours, and more preferably about 15 minutes to 1 hour. Alternatively, the temporary processing described below can be performed immediately without going through this second waiting period. In other words, the second waiting period may not be necessary.

[0047] Here, the in vitro culture is treated with the temporary treatment medium of this embodiment for only a second specified period prior to manipulation. The optimal value of this second specified period can be adjusted by those skilled in the art depending on the animal species, strain, type of manipulation, etc. Specifically, when using in vitro cultures that are less susceptible to manipulation, such as outbred strains, which are less susceptible to manipulation, the second specified period can be longer than the first specified period. For example, for unfrozen inbred mice or rats, temporary treatment with the temporary treatment medium of this embodiment can be performed for a longer period, such as 1 to 12 hours, as the second specified period. In this case, a period longer than 1 hour is more effective in suppressing embryonic development arrest. Alternatively, for pigs, temporary treatment with the temporary treatment medium of this embodiment can be performed for a longer period, such as 1 hour to 3 days, as the second specified period. Conversely, when using in vitro cultures that are sensitive to manipulation, such as inbred strains or heterologous strains, it is preferable to set the second specified period to be approximately the same as or slightly longer than the first specified period, followed by a waiting period in normal medium for the third waiting period described below. Similarly, when using frozen eggs or embryos, they are less resistant to damage caused by manipulation than unfrozen in vitro cultures, and embryonic development is more likely to be arrested by damage to DNA, etc., i.e., they are sensitive to manipulation, so it is preferable to shorten the second specified period. By configuring it in this way, the effect of suppressing the arrest of embryonic development can be further enhanced.

[0048] The manipulated explants are then harvested and transferred to normal culture medium, and optionally, a third waiting period is allowed before manipulation, such as implantation into the uterus. The cells are then allowed to develop and are obtained as developmentally engineered products, as described below.

[0049] During the specific period of treatment with these temporary treatment media, the cells are soaked in cytoskeleton regulators and / or apoptosis inhibitors, which enhance DSB repair activity in the nucleoskeleton, adjust the cell cycle to a cell cycle with high DSB repair activity, and regulate the polymerization and depolymerization of the cytoskeleton that accompanies the progression of the cell cycle, thereby suppressing pathways that would normally trigger apoptosis and cell cycle arrest due to damage, and are thought to increase resistance to damage caused by manipulation. Transferring the cells to normal medium is then thought to suppress the adverse effects of the cytoskeleton regulators and / or apoptosis inhibitors, returning them to a normal cell cycle, restoring cytoskeleton polymerization and depolymerization to normal levels, and allowing the cells to repair themselves, thereby suppressing embryonic development arrest.

[0050] The optimum value for which of the above-mentioned periods to use and the length of each period can be adjusted by those skilled in the art depending on the animal species, strain, type of operation, etc. Below are some specific examples of actual processing times:

[0051] [Table 1]

[0052] As shown in this table, for mice or rats, in the case of unfrozen inbred strains using unfrozen eggs or embryos, it is preferable that the first specified period is 30 to 1 hour, the first waiting period is 0 to 1 hour, the second waiting period is 0 to 1 hour, and the second specified period is 1 to 12 hours. Alternatively, in the case of frozen inbred strains using frozen eggs or frozen embryos, it is preferable that the first specified period is 30 to 1 hour, the first waiting period is 0 to 1 hour, the second waiting period is 0 to 1 hour, the second specified period is 30 minutes to 1 hour, and the third waiting period is 1 to 12 hours. Alternatively, in the case of inbred strains such as frozen or unfrozen wild mice or rats, or heterologous mice or rats, it is preferable that the first specified period is 30 to 1 hour, the first waiting period is 0 to 1 hour, the second waiting period is 0 to 1 hour, the second specified period is 30 minutes to 1 hour, and the third waiting period is 1 to 12 hours. In pigs, in the case of IVM and IVF embryos, it is preferable that the first specified period is 30 to 1 hour, the first waiting period is 0 hours, the second specified period is 0 hours, and the second specified period is 1 hour to 3 days.

[0053] As described above, treatment with these temporary treatment media may be performed only before the manipulation or only after the manipulation. By performing treatment with the temporary treatment medium both before and after the manipulation, it is possible to enhance the effect of suppressing embryonic development arrest, depending on the animal species, strain, and type of manipulation. In addition, when treatment with the temporary treatment medium is performed before or after the manipulation, the first specific period, first waiting period, second waiting period, and second specific period may be different, and the concentrations of each cytoskeleton regulator and / or apoptosis inhibitor contained in the temporary treatment medium may also be different. These can be optimized and adjusted by those skilled in the art. Furthermore, multiple treatments with temporary treatment medium may be performed before and / or after the procedure, in which case it is preferable to provide a waiting period after changing to normal medium after treatment.

[0054] [Method for producing developmentally engineered products, developmentally engineered products] The method for producing a developmentally engineered product of this embodiment is characterized by producing a developmentally engineered product from an in vitro culture that has been treated by the above-mentioned method for suppressing embryonic development arrest. The developmentally engineered product of this embodiment is characterized by being produced by the above-mentioned method for producing a developmentally engineered product. Here, the developmental engineering product of this embodiment includes any one or any combination of individuals, organs, tissues, and cells. Among these, individuals include chimeric individuals, model organisms, and other individuals required for experiments, reproductive engineering, and medical treatment. Organs and tissues do not necessarily have to be mature to the organ level, as long as specific differentiated cells have a specific structure as a cell mass. Furthermore, cells include dissociated cell masses without a specific structure.

[0055] The developmentally engineered products produced by this embodiment can be obtained in animals, strains, etc. that were not possible with conventional techniques, and furthermore, have few mutations due to damage, making them distinguishable to those skilled in the art. However, in the field to which the skilled artisan of this embodiment belongs, there are special circumstances in which it is very difficult for those skilled in the art to directly identify them by their structure or properties.

[0056] [Drug discovery support method] The drug discovery support method of this embodiment is characterized by evaluating a developmental engineering product produced by the developmental engineering product production method of this embodiment. In addition, the drug discovery support method of this embodiment can also administer a drug related to toxicity and / or disease for drug discovery to a developmentally engineered product, and evaluate the state of the developmentally engineered product.

[0057] Drugs related to toxicity and / or disease for drug discovery in this embodiment can be candidate drugs for drug screening that require testing for toxicity, candidate drugs for treating diseases, etc. Examples of candidate drugs in this embodiment include low molecular weight compounds, peptides, proteins, cell extracts, supernatants, fermentation products, other synthetic compounds, and natural compounds. These candidate drugs may have any purity or degree of purification. Furthermore, diseases targeted by drug screening in this embodiment include any disease. Toxicity of these can be evaluated by analyzing the expression of marker genes of the developmentally engineered products, morphological analysis, etc. Furthermore, screening may be performed in accordance with clinical trial protocols or by any method known to those skilled in the art. In these analyses, if normal function is maintained in cells administered with a candidate drug, it can be assumed that the candidate drug has low toxicity.

[0058] [Therapeutic methods, transplantation methods, medicines] The treatment method of this embodiment is a method for treating mammals, characterized by transplanting an in vitro culture treated by the above-mentioned method for suppressing embryonic development arrest and / or a developmentally engineered product produced by the above-mentioned method for producing a developmentally engineered product.

[0059] The treatment method of this embodiment can be used in reproductive medicine, for example, by applying the method for suppressing embryonic development arrest of this embodiment to frozen fertilized eggs, blastocysts, etc. This makes it possible to apply this method to mothers in whom embryo implantation is difficult due to, for example, genetic background, advanced age, various diseases, etc. In this case, by transplanting the developmentally engineered product of this embodiment into the mother's womb, the transplantation method of this embodiment also functions as a treatment method.

[0060] Alternatively, in the treatment method of this embodiment, the developmental engineering product itself produced by the above-mentioned method for producing a developmental engineering product, or a processed or extracted product, may be obtained as a pharmaceutical (medical composition) of this embodiment and used for treatment. That is, the treatment method of this embodiment can be used as regenerative medicine to treat diseases in animals, including humans.

[0061] In this treatment method of the present embodiment, the developmentally engineered product of the present embodiment is first obtained from pluripotent stem cells obtained and prepared or generated from a patient, or from a library of pluripotent stem cells with similar HLA or other types. These cells may be produced by the above-mentioned procedures, or may be produced and then manipulated, and may be treated with the above-mentioned temporary treatment medium before and / or after these procedures. The obtained pluripotent stem cells or the like are induced to differentiate and then cultured for a specific period, and the developmentally engineered product of the present embodiment is obtained at any stage, such as a cell, cell mass, tissue, or organ. The obtained developmentally engineered product may be dissociated or otherwise processed. Then, it is treated with the temporary treatment medium for any of these periods. These obtained developmentally engineered products can be used for treatment, such as injection into the site of a disease in a patient, or transplantation as at least a part of a sheet, tissue, or organ. In this case, the developmentally engineered product of this embodiment can be prepared into a single-layer or multi-layer sheet using cultureware used by those skilled in the art and transplanted into the patient. Furthermore, it is also possible to transplant a more organized culture by culturing the cells of this embodiment using an appropriate carrier or layering them using a 3D printer or the like. That is, the transplantation method of this embodiment also functions as the treatment method of this embodiment. Furthermore, the genetically engineered product of this embodiment can also be used as a medicine.

[0062] When the transplantation method and treatment method of this embodiment are applied to humans among mammals, they should be carried out to the extent and limit necessary in accordance with the ethics of various reproductive medical treatments. In other words, genetic modification, etc. should not normally be performed, and even if genetic modification, etc. is necessary for the purpose, it should be carried out to the minimum extent possible in accordance with specific standards, such as for the prevention of genetic diseases or infectious diseases. Furthermore, when the present invention is practiced in Japan, transplantation and treatment after provision of the culture will be performed by a physician. Therefore, the term "animal" in the therapeutic method of the present invention does not include humans (Homo sapiens). On the other hand, in other countries, the definitions of "animal" and "therapeutic method" are not limited.

[0063] Meanwhile, the pharmaceutical according to the embodiment of the present invention can also be used for animal therapy to treat animals other than humans. Such animals are not particularly limited and broadly include vertebrates and invertebrates. Vertebrates include fish, amphibians, reptiles, birds, and mammals. Specifically, mammals may be, for example, various animals from the aforementioned orders Primates, Rodentia, Lagomorpha, Cetartiodactyla, Perissodactyla, and Carnivora. Specific examples include mice, rats, hamsters, guinea pigs, rabbits, sheep, pigs, cows, horses, dogs, cats, ferrets, and non-human transgenic primates. Furthermore, wild animals include, in addition to mammals, fish, birds including poultry, and reptiles. Crustaceans including shrimp and insects, and other invertebrates such as squid, are also broadly included. That is, the pharmaceutical according to the embodiment of the present invention can be used not only for the treatment of humans but also for the treatment of various animals and for promoting the growth of livestock.

[0064] In addition, the pharmaceutical according to the embodiment of the present invention can be used to treat parts of the body of an animal, or organs or tissues extracted or excreted from an animal. Furthermore, this treatment is in a broad sense and can be applied to culture in bioreactors, model animals, culture of cultured organs for human transplantation, etc. In addition, the developmentally engineered product of this embodiment can be used for various therapeutic purposes other than regenerative medicine, such as the production of bioreactors, artificial organs, and the creation of cloned individuals.

[0065] When carrying out the therapeutic method according to the embodiment of the present invention, the administration interval and dosage of the developmentally engineered product can be appropriately selected and changed depending on various conditions such as the state of the disease and the condition of the subject. The single dose and frequency of administration of the genetically engineered product according to the embodiment of the present invention can be appropriately selected and changed depending on the purpose of administration, as well as various conditions such as the patient's age and weight, symptoms, and severity of the disease. The number of times and duration of administration may be just one, or administration may be once to several times a day for several weeks, and the condition of the disease may be monitored, and administration may be repeated or repeated depending on the condition.

[0066] In addition, the developmentally engineered product of the present invention can be used in combination with other compositions, etc. The developmentally engineered product of the present invention may be administered simultaneously with the other compositions, or may be administered at intervals, with no particular restriction on the order of administration. Furthermore, in the embodiments of the present invention, the period during which the disease is improved or alleviated is not particularly limited, but may be temporary improvement or alleviation, or improvement or alleviation for a certain period of time.

[0067] The above configuration can provide the following effects. Traditionally, in order to produce genetically modified animals, it has been necessary to culture embryos outside the body and manipulate them, such as by modifying their genes. However, because embryos cultured and manipulated outside the body are damaged, it is often not possible to produce individuals, depending on the strain or species. For example, when using inbred animals with conventional methods, a large number of fertilized eggs were required. Specifically, a large number of fertilized eggs were required to perform genome editing on the inbred C57BL / 6 strain using electroporation. Furthermore, when multiple in vitro embryo manipulations were performed, embryonic development almost ceased. In other words, the inbred C57BL / 6 strain could only be electroporated once. This was because the inbred C57BL / 6 strain had low resistance to damage.

[0068] Although it has been reported that mice with multiple gene mutations, floxed mice, knock-in mice, etc. can be produced by various methods, the animal species and strains that can actually be produced are limited.

[0069] Furthermore, when multiple manipulations were required, outbred strains (such as BDF1) that were resistant to damage were used. Specifically, in recent years, there have been many reports of the creation of genetically modified animals using genome editing such as CRISPR-Cas as part of basic research for the creation of disease model animals and gene therapy models. However, there have been very few reports to date other than those of specific strains of animals (e.g., mouse hybrid strains) that are highly resistant to artificial embryo manipulation. For example, with the 2STEP method, which is thought to cause the most damage due to embryo manipulation, no successful cases have been reported using pure strain mice (such as C57BL / 6) commonly used in animal experiments. Furthermore, even in hybrid mouse strains that are highly resistant to embryo manipulation, many embryos stop maturing or developing if the embryo manipulation is highly damaging, and a large supply of fertilized eggs is therefore required to reliably produce the desired genetically modified animals.

[0070] In other words, in the conventional production of genetically modified animals, it was first necessary to select animal species and strains that could withstand in vitro culture and manipulation, and then it was necessary to use a large number of these fertilized eggs and minimize damage caused by embryo manipulation.

[0071] In contrast, the temporary treatment medium according to the embodiment of the present invention reduces damage to embryos, etc. caused by in vitro culture procedures, making it possible to efficiently produce individuals, organs, tissues, cells, etc. Specifically, by treating fertilized eggs of strains with low tolerance to manipulation with a temporary treatment medium containing a cytoskeleton regulator and / or an apoptosis inhibitor, which protects and stabilizes them from damage at a specific time, embryonic development arrest can be suppressed, allowing development into blastocysts and the production of individuals. This facilitates the embryo manipulation of inbred mouse strains commonly used in experiments, reduces the number of fertilized eggs required for embryo manipulation, and increases the efficiency of individual production. In other words, it becomes possible to efficiently obtain genetically modified individuals, tissues, organs, cells, etc. from early embryos of animal species or strains that are difficult to obtain large numbers of fertilized eggs and have low tolerance to in vitro culture or manipulation. Therefore, this technology can be used as a core technology in developmental engineering to produce genetically modified animals, etc.

[0072] Specifically, as shown in the examples below, the use of a temporary treatment medium containing the apoptosis-inhibiting substance of this embodiment almost completely eliminated the embryonic development arrest that occurs after electroporation, and the normal number of fertilized eggs required for genome editing in the inbred C57BL / 6 strain was sufficient. Furthermore, the 2-step method for generating Flox mice, which previously required multiple procedures, was successfully used with the inbred C57BL / 6 strain. Furthermore, it became possible to establish ES cells and generate individual mice using xenogeneic mice, which was previously impossible.

[0073] That is, by using the temporary treatment medium of this embodiment, 1. Individuals can be produced using a small number of fertilized eggs. 2. Multiple embryo manipulations outside the body become possible. 3. Individuals and cells can be produced from various animal species and special strains using various methods such as multiple gene mutations, Flox, and knock-in. 4. It can also be applied to human assisted reproductive technology.

[0074] In the above-described embodiment and the examples described below, examples have been described in which individuals are efficiently produced as developmentally engineered products from embryos that are damaged by in vitro culture and manipulation. In this regard, it is possible to use conventional techniques for producing individuals in combination with the method for inhibiting the arrest of embryonic development, temporary treatment medium, etc. of this embodiment in order to increase the success rate.

[0075] In the above embodiment, an example was described in which a low molecular weight compound such as Y-27632 was contained in the temporary treatment medium as a Rho kinase inhibitor. However, genes, gene products, agonists / antagonists, and other compositions that act on pathways whose expression is regulated by Rho kinase can also be used as Rho kinase inhibitors.

[0076] In addition to the above-mentioned temporary treatment medium, cytochalasins and the like may also be added to the operation solution in order to prevent physical damage during operations such as cell fusion, nuclear transfer, and injection.

[0077] Furthermore, the temporary treatment medium according to the embodiment of the present invention can also be used in combination with other compositions.

[0078] Furthermore, the temporary treatment medium according to this embodiment can be used to efficiently produce individuals, organs, tissues, cells, etc. Furthermore, it is conceivable that the use of technologies other than Cas9, as shown in the following examples, can be used for genetic modification of rare breeds, gene therapy for animals with genetic diseases, treatment of pets, infertility treatment, etc. [Example]

[0079] The method for suppressing embryonic development arrest and the method for producing developmentally engineered products according to the embodiments of the present invention will be described in more detail below as examples based on specific experiments. However, these examples are merely illustrative and are not intended to limit the scope of the present invention.

[0080] Materials and Methods [reagent] (Major reagents) PZM-5 medium (Functional Peptide Institute: IFP0410P): medium for porcine embryo development, PBM medium (Functional Peptide Institute: IFP1030P): medium for porcine late embryo culture, KSOM AA medium (prepared by the present inventors), mR1ECM medium (Ark Resources), mWM medium (modified Whitten's medium) (Ark Resources), Opti-MEM medium (GIBCO BRL), M2 medium (Sigma, catalog number M7167), Y-27632 (Fujifilm Wako: 036-24023). Other IVF (in vitro fertilization)-related reagents were obtained from the Functional Peptide Institute: dbcAMP (Sigma Aldrich: D0627), FSH (Merck Serono: Gonal-F Subcutaneous Injection 150), PMSG (Kyoritsu Pharmaceutical: Ceralmon), hCG (Kyoritsu Pharmaceutical: Gestron), TGF-α (R&D Systems: 239-A), and porcine follicular fluid (prepared by the inventors; supernatant sterilized from oocyte collection solution filtered at 10,000 rpm for 30 minutes).

[0081] (Preparation of other reagents) (2nmol Alt-R CRISPR crRNA) The solution was diluted to 100 μM in 20 μL of IDTE buffer or nuclease-free water, and then aliquoted and stored at -80°C.

[0082] (20nmol Alt-R CRISPR tracrRNA) 200 μL of IDTE buffer or nuclease-free water was added to make the solution 100 μM. The solution was dispensed and stored at -80°C.

[0083] (crRNA:tracrRNA stock solution (100 μL)) The final volume was 25 μL, consisting of 4.5 μL of crRNA (100 μM) (final 18 μM), 4.5 μL of tracrRNA (100 μM) (final 18 μM), and 16 μL of Nuclease-Free Duplex Buffer. The crRNA and tracrRNA were heated at 95°C for 5 minutes and hybridized at 15–25°C. For reference, the average molecular weight of crRNA was 11,700 g / mol, resulting in a 100 μM concentration of 1.17 μg / μL. The molecular weight of tracrRNA was 22,182 g / mol, resulting in a 100 μM concentration of 2.22 μg / μL. The concentrations of crRNA and tracrRNA were 3.39 μg / μL and 33.9 ng / μL, respectively.

[0084] (Electroporation working solution (10 μL) (CUY21 EDIT)) A standard concentration was prepared by adding 1.0 μL of GeneArt Platinum Cas9 Nuclease (1 μg / μL, Thermo Scientific) (final concentration: 100 ng / μL), 1.7 μL of a 18 μM crRNA:tracrRNA stock solution (final concentration: 3 μM, approximately 100 ng / μL), 0.76 μL of 100 μM ssODN (high-concentration single-stranded oligodeoxynucleotide) (final concentration: approximately 400 ng / μL), and 6.6 μL of Opti-MEM solution.

[0085] [Equipment] The following were used: NEPA21 (registered trademark, manufactured by Nepa Gene): gene transfer device main body; CUY501P1-1.5: MS platinum block electrode, 1 mm gap capacity: 5 μL; C115CB or C115CB-2: cable (connected to the device main body); C117: cable (connected to the platinum plate electrode via C115CB).

[0086] [Methods for inhibiting embryonic development arrest, temporary treatment methods, and other culture methods] (Fertilized egg electroporation method (Technique for Animal Knockout system by Electroporation, hereinafter referred to as "TAKE") TAKE was carried out according to the method described in Patent Document 2. 1. Cas9 Nuclease, gRNA, ssODN solution, etc. were added to the electrode bathtub in a volume of genome editing solution or Opti-MEM solution appropriate for knockout or knockin. 2. The solution resistance was adjusted to 200Ω (190-210Ω). 3. First, the in vitro culture was removed from the mineral oil-covered culture medium (hereinafter referred to as "drop") and washed once with Opti-MEM solution placed in a dish. 4. The in vitro culture was then placed in the genome editing solution or Opti-MEM solution inside the electrode. 5. The actual resistance value was measured. If the resistance value was 160 Ω or less, a small amount of genome editing solution or Opti-MEM solution was aspirated, and the resistance value was measured again and adjusted to around 200 Ω (approximately 190 Ω to 210 Ω). If the resistance value was 220 Ω or more, genome editing solution or Opti-MEM solution was added, and the resistance value was measured again and adjusted to 200 Ω (approximately 190 Ω to 210 Ω). 6. After adjusting the resistance, the start button on the NEPA21 was immediately pressed to perform electroporation (EP). 7. After EP treatment, the explants were removed. Thereafter, the in vitro cultures washed with another Opti-MEM solution were placed in the liquid in the bathtub, the resistance values were made uniform, and EP was performed in the same manner.

[0087] (For unfrozen inbred strains) 1. Three hours before TAKE, five drops of Y-27632-supplemented mWM medium (final Y-27632 concentration: 10 μM; an example of the "temporary treatment medium" in this example) and Y-27632-free mWM medium (an example of the "normal medium" in this example) were placed on a 35 mm Petri dish and covered with paraffin oil to create drops. 2. Pronuclear embryos were collected from the drop, placed in mWM medium supplemented with Y-27632, and cultured for 30 minutes to 1 hour (primary treatment). 3. Then, TAKE was performed. For this TAKE, as described above, the pronuclear stage embryos were washed and then transferred to the genome editing solution or Opti-MEM solution for TAKE. The same applies to the following treatments. 4. After taking, the cells were placed in mWM medium supplemented with Y-27632 (temporary treatment) and cultured at 37°C, 5% CO2, (5% OO2) until the next day (1 hour to 12 hours). 5. If necessary, culture was continued in Y-free mWM medium or embryo transfer was performed.

[0088] (For standard frozen inbred or special strains) 1. Three hours before TAKE, five drops of Y-27632-supplemented mWM medium (final Y-27632 concentration: 10 μM; an example of the "temporary treatment medium" in this example) and Y-27632-free mWM medium (an example of the "normal medium" in this example) were placed on a 35 mm Petri dish and covered with paraffin oil to create drops. 2. Pronuclear embryos were collected from the drop and cultured in mWM medium containing Y-27632 for 30 minutes to 1 hour (primary treatment). 3. Then, TAKE was performed. 4. After taking, the cells were placed in mWM medium supplemented with Y-27632 and cultured for 30 minutes to 1 hour (primary treatment). 5. The embryos were then placed in Y-free mWM medium and cultured at 37°C, 5% CO2, and 5% O2 until the next day. If necessary, the embryos were cultured in Y-27632-free mWM medium or transferred.

[0089] (For frozen and unfrozen inbred strains derived from heterologous wild mice) 1. Three hours before TAKE, five drops of Y-27632-supplemented mWM medium (final Y-27632 concentration: 10 μM; an example of the "temporary treatment medium" in this example) and Y-27632-free mWM medium (an example of the "normal medium" in this example) were placed on a 35 mm Petri dish and covered with paraffin oil to create drops. 2. Two-cell stage embryos were collected or thawed from the drop and cultured in mWM medium supplemented with Y-27632 for 30 minutes to 1 hour (primary treatment). 3. Then, TAKE was performed. 4. After the take-up, the cells were placed in mWM medium supplemented with Y-27632 and cultured for 30 minutes to an hour (primary treatment). They were then placed in mWM medium without Y-27632 and cultured at 37°C, 5% CO2, and 5% O2 until transplantation. 5. After that, the culture was continued in mWM medium without Y-27632 as needed to establish ES cells.

[0090] (Pig IVM, IVF embryos) 1. On the day before the take, five drops of Y-27632-supplemented PZM-5 medium (Y-27632 final concentration 10 μM, an example of the "temporary treatment medium" in this example) were placed on a 35 mm Petri dish and covered with paraffin oil to create drops. For the drops in this example, 20 μL of medium was added, followed by 1 mL of oil, and then 20 μL of medium was added again and completely covered with oil. Twice as many of these drops were prepared as were those to be taken. 2. IVF was performed according to techniques common to those skilled in the art. 3. The day after insemination, the IVF embryos were placed in PZM-5 medium supplemented with Y-27632 and cultured for 30 minutes to 1 hour (primary treatment). 4. Then, TAKE was performed. 5. The cells were returned to PZM-5 medium without Y-27632 (an example of the "normal medium" in this example) and cultured at 39°C, 5% CO2, and 5% O2. 6. If necessary, sampling was performed at the developmental stage. If culture was to continue until the blastocyst stage, drops containing PZM-5 medium and PBM medium without Y-27632 were prepared in the same manner as in step 1 four days after insemination. 7. Five days after insemination, embryos that had developed to the morula and blastocyst stages were transferred to PBM medium (an example of "normal medium" in this example). The remaining surviving embryos were also transferred to PZM-5 medium without Y-27632, and as development progressed, they were transferred to PBM medium as appropriate. 8. Seven to eight days after insemination, the embryos developed into blastocysts and expanded blastocysts.

[0091] (Inbred mouse 2-step method) 1. Three hours before the TAKE, five drops of Y-27632-supplemented mWM medium (final concentration of Y-27632: 10 μM, an example of the "temporary treatment medium" in this example) and Y-27632-free mWM medium (an example of the "normal medium" in this example) were placed on a 35 mm Petri dish and covered with paraffin oil to create drops. The number of drops prepared was twice the number of drops to be taken. 2. Pronuclear embryos were collected and cultured in mWM medium containing Y-27632 for 30 minutes to 1 hour (primary treatment). 3. After that, the first take was done. 3a. In the case of frozen eggs, after the first take, they were placed in mWM medium supplemented with Y-27632 and cultured for approximately 30 minutes to 1 hour (primary treatment). 3b. In the case of unfrozen eggs, after the first take, they were placed in mWM medium supplemented with Y-27632 and cultured at 37°C, 5% CO2, (5% O2) conditions until the next day (temporary treatment). 4. After the initial treatment, the cells were placed in mWM medium without Y-27632 and cultured at 37°C, 5% CO2, (5% O2) until the next day. 5. In both cases, the embryos were checked the next day, and a second TAKE was performed on the embryos that had reached the two-cell stage. 6. After the second TAKE, the cells were placed in mWM medium supplemented with Y-27632 and cultured for 30 minutes to 1 hour (primary treatment). 7. The embryos were then transferred to mWM medium without Y-27632. If necessary, the culture was continued or embryo transfer was performed.

[0092] (Inbred rat 2-step method) 1. Three hours before TAKE, five drops of Y-27632-supplemented KSOM AA medium (Y-27632 final concentration 10 μM, an example of the "temporary treatment medium" in this example) were placed on a 35 mm Petri dish and covered with paraffin oil to create drops. 2. Pronuclear embryos were collected and cultured in KSOM AA medium supplemented with Y-27632 for 30 minutes to 1 hour (primary treatment). 3. After that, the first take was done. 4. After taking, the cells were placed in KSOM AA medium supplemented with Y-27632 and cultured at 37°C under 5% CO2 and 5% O2 conditions for 30 minutes to 12 hours (temporary treatment). 5. Thereafter, the cells were placed in KSOM AA medium without Y-27632 (an example of the "normal medium" in this example) and cultured at 37°C, 5% CO2, and 5% O2 until the second take. 6. The next day, the two-cell embryos were separated and, approximately 20 to 22 hours after egg collection, were placed back into KSOM AA medium supplemented with Y-27632 and cultured for approximately 30 to 1 hour (primary treatment). 7. After that, a second take was done. 8. After the second TAKE, the cells were placed in KSOM AA medium supplemented with Y-27632 and cultured for 30 minutes to 1 hour (primary treatment). 9. The embryos were then transferred to mR1ECM medium (an example of the "normal medium" in this example). If necessary, the culture was continued or embryo transfer was performed.

[0093] (embryo transfer) According to standard methods, mice and rats were mated with vasoligated males, and in the afternoon after plugs were confirmed, approximately 10 embryos were implanted into the left and right oviducts or uterus of mature female mice and rats with confirmed plugs. In the case of pigs, around 10 embryos were transferred non-surgically into the uterus.

[0094] 〔result〕 [Overall summary] First, referring to FIG. 2, the strains and procedures that made it possible to produce individuals by the method for suppressing embryonic development arrest and the method for producing developmentally engineered products of this example will be described. Figure 2 shows the degree of difficulty in generating individuals for crossbreed strains, closed colonies, inbred strains (e.g., B6), disease models, and xenogeneic mice. For rats, the degree of difficulty in generating individuals for crossbreed strains, closed colonies, inbred strains (e.g., F344), and disease models is shown. The degree of difficulty in generating individuals for pigs, sheep, dogs, monkeys, and humans is also shown. For each procedure, procedures that damage embryos (cells) are shown, including superovulation, frozen embryos (transplantation, embryo freezing), nuclear transfer (NT), intracytoplasmic sperm injection (ICSI), microinjection (MI), and electroporation (EP). Furthermore, procedures that damage the nucleus, such as exposure to large amounts or high-molecular-weight DNA and procedures involving multiple DSBs (e.g., 2-step DNA synthesis), are shown. Furthermore, procedures that create aneuploids and polyploids (e.g., tetraploids) through cell fusion are shown. Specifically, the symbols in each column indicate the difficulty of creating an individual: "◎" (easy, low difficulty), "〇" (possible, normal difficulty), "△" (high difficulty), and "×" (impossible). In other words, the order of difficulty is "◎" - "〇" - "△" - "×". "?" indicates that the difficulty is unknown. Here, the difficulty level at the end of the arrow in each column indicates what has actually been demonstrated by the inventors to be possible to produce using the method for inhibiting embryonic development arrest and the method for producing developmentally engineered products of this example. The areas with a gray (dark) background in each column indicate areas where the effects of this example can be expected, but where no test examples have yet been performed. Thus, the method for suppressing embryonic development arrest and the method for producing developmentally engineered products of this example can be implemented for a wide range of mammals, lineages, and manipulations.

[0095] Below, examples of treatment of each in vitro culture shown in FIG. 2 will be explained.

[0096] (Processing example 1) First, an example of producing a knockout mouse by electroporation using a C57BL / 6J mouse will be described. Conventionally, fertilized eggs treated with electroporation (TAKE) have low development and birth rates, so it is necessary to increase the number of embryos transferred per recipient. In this example, the addition of Y-27632 was aimed at increasing the development rate and birth rate and reducing the number of embryos to be transferred. For this reason, in this production example, the embryos were temporarily treated with a temporary treatment medium containing Y-27632 before and after TAKE. For in vitro culture, pronuclear stage embryos of C57BL / 6J (Claire Japan), MCH (female, manufactured by Claire Japan), and female embryos were used.

[0097] As shown in Figure 3, the results were obtained by culturing the cells in a culture medium containing Y-27632, an example of the cytoskeleton regulator and / or apoptosis inhibitor of this embodiment, before and after electroporation. When the Mk-3 gene was knocked out using two types of gRNA ("MKIII ver. 1" or "MKIII ver. 2"), the birth rate increased approximately fivefold. The efficiency of non-homologous end joining (NHEJ) remained unchanged.

[0098] (Processing example 2) Next, an example of producing Flox mice by the 2STEP method using C57BL / 6J will be described. In the past, inbred fertilized eggs treated with the 2STEP method had significantly lower development and birth rates, making it impossible to obtain inbred Flox mice. Therefore, in this production example, we aimed to increase the development and birth rates and produce inbred Flox mice. For this reason, in this production example, the embryos were temporarily treated with a temporary treatment medium containing Y-27632 before and after electroporation (EP) using the 2-step method. Pronuclear stage embryos (MCH) (female, manufactured by Claire Japan) of C57BL / 6J (Claire Japan) were used as in vitro culture materials.

[0099] The present inventors conducted an experiment to obtain Flox mice using the 2STEP method targeting the Mecp2 gene. The results are shown in Table 2 below:

[0100] [Table 2]

[0101] In Table 2, "m" indicates treatment with normal medium, and "Y" indicates temporary treatment with temporary treatment medium. Each experimental group was treated with normal medium (m) or temporary treatment medium (Y) in the first electroporation step and the second electroporation step of the 2-STEP method, respectively. That is, "m->m" indicates all treatments in normal medium, "m->Y" indicates temporary treatment with temporary treatment medium only in the first electroporation step, and "Y->Y" indicates temporary treatment with temporary treatment medium in both steps. Each column shows the percentage of cells in which the loxP sequence was inserted only on the left side by the first electroporation step (Left loxP), cells in which the loxP sequence was inserted only on the right side by the second electroporation step (Right loxP), and cells in which both loxP sequences were inserted by both steps (2loxP).

[0102] As a result, one out of 15 blastocysts (7%) and one out of 25 newborns (4%) of C57BL / 6J Flox mice were obtained. On the other hand, in the experimental group without Y-27632, neither blastocysts nor newborns were obtained.

[0103] Similarly, newborn C57BL / 6J floxed mice were obtained for the Drb1 gene. An example of a Flox mouse in which conditional knockout was possible with 2loxP is shown in Figure 4. In lane "27," a band (5398 bp or 5440 bp) indicating that it is a Flox mouse is generated.

[0104] In this way, we attempted to create inbred Flox mice using two genes that could be conditionally knocked out using the 2STEP method, and in both cases, we obtained inbred Flox mice only from the experimental group that received Y-27632, a type of mouse that had not previously been reported to be successful. Thus, we believe that temporary treatment with Y-27632 is effective in strains that are sensitive to damage, such as B6.

[0105] (Processing example 3) Next, we will explain an example of producing individuals from frozen eggs of the inbred strain SPR2 of the heterologous mouse Mus spretus (Algerian house mouse) (hereinafter simply referred to as "heterologous inbred mouse SPR2" or simply "SPR2"), and an example of the process for establishing ES cells from frozen embryos of a heterologous mouse. In this production example, two-cell frozen eggs of SPR2 were thawed, cultured in a temporary treatment medium containing Y-27632 for 1 hour, and then transplanted into the uterus of a pseudopregnant female mouse. In this example, two-cell frozen eggs from the heterologous inbred mouse strain SPR2 were thawed, cultured for one hour in a culture medium containing Y-27632, and then transplanted into the uterus of a pseudopregnant female mouse. As a result, individuals derived from the frozen eggs (16 transplanted embryos and 6 liveborn offspring) were obtained. The results are shown in Table 3 below:

[0106] [Table 3]

[0107] An example of an individual obtained is shown in Figure 5. It has previously been impossible to create an individual from frozen embryos of the heterologous inbred mouse strain SPR2.

[0108] Next, the results of an example of the establishment of ES cells from frozen embryos of the heterologous inbred mouse strain SPR2 will be described. After thawing two-cell frozen eggs from the heterologous inbred mouse strain SPR2, they were cultured to the blastocyst stage in a temporary treatment medium containing Y-27632, and attempts were made to establish ES cells. As a result, ES cells were established efficiently (five frozen embryos, two established lines). On the other hand, without the addition of Y-27632, individuals and ES cells were not established. Thus, temporary treatment with Y-27632 made it possible to create ES cells from frozen embryos of different mouse species, something that had previously been impossible.

[0109] (Processing example 4) Next, an example of the treatment for establishing ES cells from electroporated early embryos of the heterologous inbred mouse strain SPR2 and the heterologous mouse Mus caroli (Okinawa house mouse) will be described. We cultured early embryos of the heterologous inbred mouse strain SPR2 and the heterologous mouse strain Mus caroli in a medium containing Y-27632, then electroporated them to attempt genome editing, resulting in the establishment of ES cells with a high success rate. Genome editing was performed on Mus caroli ES cells, and analysis of some of the cells demonstrated that NHEJ had occurred, allowing the establishment of heterologous mouse ES cells. Figure 6 shows a dish of ES cells established in Mus caroli. The morphology of ES cells can be visually confirmed. Figure 7 shows Mus caroli with a Tyr gene knockout. The arrowhead indicates the band representing the knockout.

[0110] (Processing example 5) Next, an example will be described in which it was verified whether the temporary treatment medium of this example can reduce the adverse effects on the occurrence of electroporation. First, we investigated the effects on development after electroporation using inbred rat embryos. Inbred rat strain F344, embryonic development arrest occurs when electroporated twice. Regarding this, the results were confirmed by culturing the rats in the temporary treatment medium of this embodiment for specific periods before and after electroporation.

[0111] 8A shows the results of a control that was not treated with the temporary treatment medium of this embodiment. None of the embryos developed to the blastocyst stage. Figure 8B shows the results of treatment with the temporary treatment medium of this embodiment before and after electroporation, with 71.8% developing to the blastocyst stage. Figure 8C shows a control that was not electroporated or treated with the temporary treatment medium of this embodiment, and 71.4% of the embryos developed to the blastocyst stage. In these figures, the incidence rate of 2-cell stage embryos was calculated as the ratio of 2-cell stage embryos to pronuclear stage embryos, and the incidence rate of blastocyst stage embryos was calculated as the ratio of blastocyst stage embryos to 2-cell stage embryos.

[0112] As a result, when the inbred rat strain F344 was treated with the temporary treatment medium of this embodiment before and after electroporation, embryos developed to the blastocyst stage without arresting their development. Specifically, 74 of 103 embryos at the 2-cell stage (71.8%) developed to the blastocyst stage. This rate was almost the same as that of the untreated control without electroporation.

[0113] Next, the effect of the temporary treatment medium of this example was measured in the heterogeneous inbred mouse strain SPR2, which cannot be used to obtain individuals by embryo transfer after freeze-thawing and electroporation. The results are shown in Table 4 below:

[0114] [Table 4]

[0115] Thawed 2-cell stage embryos were cultured in the temporary treatment medium of this example for a specific period before and after electroporation. Seventeen 2-cell stage embryos of the heterologous inbred mouse strain SPR2 were transplanted into the oviducts, yielding seven individuals. Furthermore, 16 embryos (blasts) developed to the blastocyst stage in mWM medium were transplanted into the uterus 3.5 days after plug confirmation, and five individuals were obtained.

[0116] Furthermore, the effect of the temporary treatment medium of this example was measured on porcine fertilized embryos that had been matured in vitro and fertilized in vitro using frozen semen. As a result, when cultured in the temporary treatment medium of this example, 35 of 123 2-cell stage embryos developed into late blastocysts (expanded or erupted blastocysts). In contrast, when cultured in the control normal medium only and electroporated, no blastocysts were obtained.

[0117] (Processing example 6) Next, an example of generating Flox rats by electroporation-mediated knock-in (KI) using high-concentration ssODN in the inbred rat strain F344 will be described. When inbred F344 rats were electroporated, increasing the ssODN concentration and number of translation pulses resulted in developmental arrest. Furthermore, even with the normal concentration and number of pulses, developmental arrest occurred after two electroporations. Inbred F344 rats were cultured in the temporary treatment medium of this example before and after electroporation to verify whether increasing the ssODN concentration and number of translation pulses would result in knock-in of the LoxP sequence that had not been knocked in, and whether two-step Floxing was possible.

[0118] In this example, knock-in was performed using the 2STEP method in F344 rat pronuclear stage embryos. Electroporation was performed with 800 ng / μL ssODN, twice the usual concentration, and 14 transfer pulses, approximately twice the usual concentration. The embryos were cultured in the temporary treatment medium described in this example for specific periods before and after electroporation. The results are shown in Table 5 below:

[0119] [Table 5]

[0120] Of the 86 samples shown in the table, 67 were selected that had developed to the blastocyst stage, and those with the Left LoxP sequence knocked in were first examined by PCR. When treated with normal medium alone, LoxP knock-in was not possible (0 / 12, 0%), whereas it was confirmed in the blastocyst stage when treated with the temporary treatment medium of this example (19 / 67, 28%). Furthermore, the embryos developed to the blastocyst stage without developmental arrest, and Flox, which has loxP sequences at both ends of the target gene, was confirmed in the blastocyst stage (1 / 67, 1.5%). In other words, the LoxP sequence, which could not be knocked in using the normal ssODN concentration and number of pulses, could be knocked in by culturing in a culture medium containing Y-27632 before and after electroporation. In this case, it was possible to double the ssODN concentration during electroporation and double the number of electroporations.

[0121] (Processing example 7) Next, we will explain an example of producing multiple gene knockout (Knock-Out, KO) mice from a small number of fertilized embryos from mice for which it is difficult to obtain a large number of fertilized eggs, such as disease model mice. In this example, we created multiple gene knockout mice using immunodeficient nude mouse embryos. Specifically, pronuclear embryos were collected from a BALB / c-nu / nu male mated with a superovulated BALB / c-nu / + female. Two guide RNAs (crRNA:tracrRNA) for the BBOX1 gene and two for the IL2RG gene were electroporated at approximately 100 ng / μL per embryo (total of approximately 400 ng / μL) with 14 transfer pulses. The embryos were then cultured in the temporary treatment medium described in this example before and after electroporation. As a result, 63 embryos were transferred and 16 pups were born. Analysis of some of the pups (4 live births and 3 stillborns) revealed that mutations were found in 3 of them, and mutations in two genes were confirmed in 1 of them. #2 and #4, the live births in Table 6 below, are successful cases.

[0122] [Table 6]

[0123] As a result, we were able to generate nude mice in which both the mBBOX1 and mIL2RG genes were knocked out, whereas control mice treated with normal medium alone did not produce offspring.

[0124] (Processing example 8) Next, an example of producing a heterologous knockout (KO) mouse will be described. Two-cell embryos of the heterologous inbred mouse strain SPR2 were electroporated once or twice with four guide RNAs (crRNA:tracrRNA) targeting the Tyr gene at approximately 100 ng / μL per embryo, for a total of approximately 400 ng / μL. Before and after electroporation, the embryos were cultured in the temporary treatment medium described in this example. The results are shown in Table 7 below:

[0125] [Table 7]

[0126] As a result, when electroporation was performed only once, 20 embryos were transferred and 7 individuals were obtained, of which 3 were mosaics. When electroporation was performed twice, 13 embryos were transferred and 7 individuals were obtained, of which 3 were mosaics and 3 were white (homozygous or compound heterozygous). 9A, 9B, and 9C show examples of the obtained individuals.

[0127] Thus, we succeeded in generating knockout mice with the Tyr gene knocked out from two-cell frozen eggs of the heterologous inbred mouse strain SPR2. In other words, we were able to efficiently generate KO homozygotes (including compound heterozygotes) using heterologous inbred mouse strains.

[0128] (Processing Example 9) Next, an example will be described in which early embryos of the inbred mouse B6J strain were used, and frozen embryos stored at the blastocyst stage were used to compare whether development was affected by using the normal medium temporary treatment medium of this embodiment before and after manipulation.

[0129] In this treatment example, the temporary treatment medium used was mWM medium supplemented with Y-27632 at a concentration of 10 μM (referred to as "Y+ medium" in this example). The normal medium used was mWM medium without Y-27632 (referred to as "Y- medium" in this example).

[0130] For the treatment, the prepared frozen embryos were thawed and allowed to develop directly in Y-medium until the blastocyst stage. Next, half of the embryos that had developed to the blastocyst stage were transferred to Y+ medium and left there for 30 minutes, after which the embryos were transferred back to Y- medium and left there for 30 minutes. Then, embryos treated with Y+ medium and embryos not treated with Y+ medium were frozen in one tube each. Both tubes were then thawed and placed in normal medium for 10 minutes. Thereafter, the embryos were equally divided into Y+ medium and Y- medium, and placed for 1 hour. Next, each tube was returned to Y-medium and the embryos were observed immediately after thawing, 3 hours later, and 24 hours later.

[0131] The results of these operations and treatments are shown in Table 8 below.

[0132] [Table 8]

[0133] Comparing Y- and Y+ medium, we found that more embryos survived and recovered in Y+ medium. Embryos using Y+ medium before and after freezing had the best recovery rate, followed by those using Y+ medium before freezing and then Y- medium after freezing. This was followed by those using Y- medium, Y+ medium before and after freezing, and those left in Y- medium. Although each embryo had been frozen twice, 92% recovered when developed in Y+ medium.

[0134] (Processing example 10) Next, an example will be described in which it was verified whether cytochalasin B (hereinafter referred to as "CB"), which is an intracellular skeleton regulator other than Y-27632, has a similar effect. In this treatment example, normal medium (hereinafter referred to as "Y- medium" as in Treatment Example 9), temporary treatment medium supplemented with Y-27632 (hereinafter referred to as "Y+ medium" as in Treatment Example 9), and medium supplemented with CB (hereinafter referred to as "CB+ medium") were used, as in Treatment Example 9. CB+ medium was prepared with CB concentrations of 5 μM and 10 μM. The recovery rates were compared when frozen embryos were thawed and developed, then refrozen as blastocysts, and then thawed again.

[0135] For the treatment, 107 frozen 2-cell stage C57BL / 6J (manufactured by Claire) embryos were used. First, the frozen 2-cell stage embryos were thawed and then allowed to develop into blastocysts in a normal medium. Next, the blastocysts were transferred to each of the above media and left for 30 minutes, after which the embryos were transferred again to Y-medium and left for 30 minutes. The embryos were then individually frozen. The embryos were then thawed and placed in M2 medium for 10 minutes. Then, the embryos were evenly distributed among the culture media and placed on the media for 1 hour. The embryos were then returned to Y-medium and observed immediately after thawing, 3 hours later, and 24 hours later.

[0136] The results of these operations and treatments are shown in Table 9 below. [Table 9]

[0137] As a result, when CB+ medium and Y+ medium were compared, it was confirmed that the CB+ medium, like the Y+ medium, resulted in a high rate of embryo survival and recovery. Specifically, a comparison of Y- medium (control), Y+ medium, and CB+ medium revealed that the medium containing Y-27632 provided the greatest embryo survival and recovery. The next best result was CB+ medium (CB concentration 5 μM). Data observed 3 hours after CB+ (CB concentration 5 μM) showed results comparable to those of Y+ medium. Comparing the concentrations of CB+ medium, the recovery rate was 15% higher at 5 μM than at 10 μM after 24 hours. The control Y-medium had the lowest recovery rate among the tested media.

[0138] It goes without saying that the configurations and operations of the above-described embodiments are merely examples, and can be modified as appropriate within the scope of the present invention. [Example]

[0139] Next, as Example 2 of the present invention, the results of a treatment example in which in vitro cultures damaged by other strains or other types of manipulations were temporarily treated will be described. For each treatment example, the materials and methods were the same as in Example 1 above.

[0140] The inbred mouse strain B6J, the combined immunodeficient mouse strain NOD-scid, and the inbred rat strain Wistar-Imamichi according to Example 2 were all manufactured by CLEAR Japan.

[0141] (Processing example 11) Using mice of an inbred strain (B6J of the B6 strain) different from the inbred mice of Treatment Example 2 in Example 1, knock-in was performed by the same two-step method as in Treatment Example 2 above, and further individuals were produced. The results of individual production using this 2-STEP method are shown in Table 10 below.

[0142] [Table 10]

[0143] Furthermore, similarly, we also produced individuals from the inbred B6J strain of mice using the electroporation method (1STEP method) with high-concentration ssODN. The results are shown in Table 11 below.

[0144] [Table 11]

[0145] Thus, it became possible to produce Flox inbred mice using the 1STEP method.

[0146] (Processing example 12) Pronuclear embryos of the same inbred rat strain as in Treatment Example 6 in Example 1 were subjected to knock-in by electroporation (1STEP method) using high-concentration ssODN, and then individuals were produced. As in the above-mentioned Treatment Example 6, LoxP, which was not knocked in using a normal ssODN concentration and number of transfer pulses, was subjected to the same temporary treatment as in the above-mentioned Example 1. Specifically, before and after electroporation, a 1-hour temporary treatment, 24 hours of culture in normal medium (waiting period), and another 1-hour temporary treatment were performed. This enabled us to double the ssODN concentration and the number of electroporations, resulting in knock-in. Furthermore, by increasing the Cas9 concentration five-fold, we were able to obtain Flox inbred rats with a single electroporation. The results are shown in Tables 12 and 13 below.

[0147] [Table 12]

[0148] [Table 13]

[0149] As a result, we were able to simultaneously insert Flox sequence DNA fragments into multiple sites in early inbred rat embryos, which are extremely difficult to manipulate, and create individuals after genome editing by doubling the gRNA concentration and increasing the Cas9 protein concentration by five times.

[0150] In this way, it was possible to simultaneously edit multiple regions of the early embryos of inbred rats and mice, which are extremely difficult to manipulate.

[0151] (Processing example 13) We generated homozygous large deletion knockout individuals using inbred rats by electroporation. When the Klotho gene (40 Kbp), which cannot normally be deleted, was knocked out by electroporation in the inbred Wistar-Imamich strain and the inbred WKY strain, a temporary treatment similar to that described in Example 1 was performed. Specifically, before and after electroporation, a 1-hour temporary treatment, 24 hours of culture in normal medium (waiting period), and another 1-hour temporary treatment were performed. This resulted in highly efficient knockout inbred rat strains lacking a gene of 40 kbp (large deletion), most of which were homozygous. The results are shown in Tables 14 and 15 below.

[0152] [Table 14]

[0153] [Table 15]

[0154] An example of an individual generated as a large deletion knockout inbred rat is shown in Figure 10. "With large deletion" is the large deletion knockout inbred rat of this example, and "without large deletion" is the control rat. In this way, it became possible to create homozygous individuals with large gene deletions.

[0155] As described above, in this embodiment, it is possible to produce genome-edited individuals of inbred mice, inbred strains of heterogeneous mice, inbred rats, or rats equivalent to inbred strains by multiple electroporations, and to produce genome-edited individuals of inbred mice, inbred strains of heterogeneous mice, inbred rats, or rats equivalent to inbred strains by introducing high concentrations of DNA, RNA, or protein into fertilized eggs.

[0156] (Processing example 14) Next, similarly to Treatment Example 7 of Example 1, genome editing of multiple genes was performed on a different strain of severe combined immunodeficient mice, the NOD-scid strain, to produce individuals. Here, NOD-scid mice in which both the mBBOX1 and mIL2RG genes were knocked out were produced using the same treatment method as in the above-mentioned treatment example used on BALB / c nude mice. The results are shown in Table 16 below.

[0157] [Table 16]

[0158] As a result, 6 out of 9 individuals (66.7%) had mutations in one of the two genes. In this way, it has become possible to perform genome editing on multiple sites in multiple genes in immunodeficient early mouse embryos, which are extremely difficult to manipulate and not easy to obtain (prepare), and to create individuals.

[0159] (Processing example 15) Next, in the same manner as in Treatment Example 9 of Example 1, early embryos of the inbred mouse strain B6J, rather than normal mice, were subjected to multiple freeze-thaw cycles to examine whether they could be recovered. The results are shown in Table 17 below.

[0160] [Table 17]

[0161] (Processing example 16) Next, knockouts were also produced by electroporation in unfrozen pronuclear embryos (1 cell) of the inbred rat strain F344 in the same manner as in Treatment Example 1 of Example 1. The conditions were a gRNA concentration twice the specified amount, 14 pulses, and a reduction rate of 20%. The results are shown in Table 18 below.

[0162] [Table 18]

[0163] (Processing example 17) Next, we generated knockouts using TAKE in pronuclear (1-cell) and 2-cell (2-cell) embryos of the inbred rat strain Wistar-Imamichi. The conditions were a fixed gRNA concentration (1x), 7 pulses, and a reduction rate of 40%. The results are shown in Table 19 below.

[0164] [Table 19]

[0165] In this way, even inbred mice and rats can be subjected to highly damaging manipulations, particularly those involving multiple freeze-thaw cycles at the two-cell stage, in addition to pronuclear stage embryos.

[0166] (Processing example 18) Next, we investigated the developmental efficiency of frozen embryo transfer using blastocysts from C57BL / 6J mice. Specifically, C57BL / 6J mouse blastocysts were frozen and thawed using either the Y-27632-treated medium or a control standard medium. An average of 20 blastocysts per mouse were then transferred into the uterus of recipient MCH mice. The mice were delivered by Caesarean section (CS), and implantation scars and the number of offspring were confirmed. The mice were then suckled by foster mothers, and the number of weaned mice was counted, and the weaning survival rate was calculated. The embryo culture medium used was based on mWM medium. The results are shown in Table 20 below.

[0167] [Table 20]

[0168] As a result, when frozen blastocysts were thawed and transferred using Y-27632, the number of offspring born per number of transferred blastocysts was higher and the weaning survival rate was also improved compared to when Y-27632 was not used. Therefore, the temporary treatment medium containing Y-27632 according to this embodiment was able to reduce damage to embryos during freezing and thawing of blastocysts and stabilize them.

[0169] (Processing Example 19) Next, we examined the efficiency of multi-target genome editing using early embryos of the immunodeficient mouse NOD-scid. High-concentration RNA-protein mixtures containing CRISPR-Cas9 at multiple sites (four sites in total) in multiple genes were electroporated (genome-edited) into early embryos of immunodeficient mice, which are extremely difficult to manipulate and difficult to prepare, and the embryos were then transplanted into the recipient uterus, resulting in offspring. The results are shown in Table 21 below.

[0170] [Table 21]

[0171] As a result, no offspring were obtained when the temporary treatment medium containing Y-27632 according to the present embodiment was not used. On the other hand, when Y-27632 was used, offspring were obtained even with a small number of implanted eggs, withstanding the stress of high concentrations of RNA-protein and electroporation due to genome editing. Furthermore, the efficiency of genome editing was dramatically improved compared to conventional methods. Thus, by using a temporary treatment medium containing Y-27632 according to this embodiment, (1) early embryos of immunodeficient mice, which are difficult to manipulate, can be stabilized and offspring can be efficiently obtained. (2) The stress of high-concentration nucleic acid-protein solutions and electroporation can be avoided. (3) The repair efficiency of double-strand breaks (DSBs) caused by genome editing can be increased, resulting in highly efficient genome editing.

[0172] Nude mice, disease model inbred rats, and mice generally have low reproductive capacity. The number of fertilized eggs that can be collected is small, and it is difficult to manipulate fertilized eggs in vitro. The same is likely true for rare breeds and endangered species. By performing various treatments using the temporary treatment medium according to this embodiment, it becomes possible to utilize breeding and reproductive engineering using these rare, precious, and fragile early embryos.

[0173] (Processing example 20) Next, the passage efficiency of mice produced using the temporary treatment medium according to this embodiment was examined. The NOD-scid mice of Treatment Example 14 described above were used to crossbreed IL2RG gene and Bbox1 gene double knockout mice with each other, and it was examined whether Y-27632 had any effect on the offspring mice. The results are shown in Table 22 below.

[0174] [Table 22]

[0175] As a result, an average of 6-8 pups were born per litter in both the first generation (F1) and second generation (F2). This is almost the same as the birth rate of the NOD-scid mice, the base breeding model. Furthermore, the offspring mice showed no abnormalities in phenotype. Thus, the use of Y-27632 according to this embodiment did not affect the passage and phenotype of the mice.

[0176] Here, commercially available "NOG" strain mice are severely immunodeficient mice in which the IL2RG gene has been disrupted in NOD-scid mice. Because breeding of these commercially available NOG mice is not permitted under license, it is not possible to freely create combination mice with knockout mice for other genes. Even in such cases, direct generation of double knockouts including IL2RG makes them easily suitable for use in research. [Industrial Applicability]

[0177] INDUSTRIAL APPLICABILITY The present invention can provide a temporary treatment medium for reducing damage caused by manipulation to in vitro cultures, and is industrially applicable.

Claims

1. A temporary treatment medium for reducing damage caused by manipulation to an in vitro culture containing any one or any combination of germ cells, fertilized eggs, and embryos other than primordial germ cells, comprising: containing a cytoskeleton modulating agent and / or an apoptosis inhibitor, the cytoskeleton regulator and / or the apoptosis inhibitor is a Rho kinase inhibitor; The operation is a treatment that causes damage to the in vitro culture, excluding freezing and thawing operations, The damage involves damage or alteration of various structures necessary for cell survival and normal differentiation, and double-strand breaks in DNA within the nucleus. A temporary treatment medium characterized by:

2. The Rho kinase inhibitor is Contains Rock inhibitors The temporary treatment medium according to claim 1 .

3. the Rock inhibitor is Y-27632; The concentration of the Y-27632 is 0.1 μM to 20 μM.

3. The temporary treatment medium according to claim 2.

4. The in vitro culture is from the order Primates, Rodents, Lagomorpha, Cetartiodactyla, Perissodactyla, or Carnivora.

4. The temporary treatment medium according to claim 1, wherein the primary treatment medium is a medium containing a soluble component.

5. The manipulation may include any one or any combination of superovulation treatment, nuclear transfer, intracytoplasmic sperm injection, and introduction of DNA or RNA into the mother. The temporary treatment medium according to claim 1 .

6. The damage includes damage caused by physical or organic stress on cells, or damage caused by reactive oxygen species or chemicals associated with ionization or destruction of intracellular organelles.

6. The temporary treatment medium according to claim 1 or 5.

7. The manipulation includes a reproductive medical treatment of the in vitro culture.

7. The temporary treatment medium according to any one of claims 1, 5 and 6.

8. The temporary treatment medium according to any one of claims 1 to 7 is included. A processing kit comprising:

9. Contains an apoptosis inhibitor, Reduces damage caused by manipulation of in vitro cultures and inhibits embryonic arrest. The in vitro culture comprises any one or any combination of germ cells excluding primordial germ cells, fertilized eggs, and embryos, the cytoskeleton modulating agent and / or apoptosis inhibitor is a Rho kinase inhibitor; The operation is a treatment that causes damage to the in vitro culture, excluding freezing and thawing operations, The damage involves damage or alteration of various structures necessary for cell survival and normal differentiation, and double-strand breaks in DNA within the nucleus. An agent for inhibiting embryonic development arrest.

10. The embryonic development arrest inhibitor is for use in a temporary treatment medium. The embryo development suppression arresting agent according to claim 9.

11. The Rho kinase inhibitor is Contains Rock inhibitors The embryonic development arrest inhibitor according to claim 9 or 10.

12. The Rock inhibitor is Y-27632. The embryonic development arrest inhibitor according to claim 11.

13. The manipulation may include any one or any combination of superovulation treatment, nuclear transfer, intracytoplasmic sperm injection, and introduction of DNA or RNA into the mother. The embryo development suppression arresting agent according to claim 9.

14. A method for suppressing embryonic development arrest, which reduces damage caused by manipulation to an in vitro culture containing any one or any combination of germ cells excluding primordial germ cells excluding humans (Homo sapiens), fertilized eggs excluding humans, and embryos excluding humans, and suppresses embryonic development arrest, comprising: treating the cells with a temporary treatment medium containing a cytoskeleton modulating agent and / or an apoptosis inhibitor for a specific period before and / or after the damaging operation; the cytoskeleton regulator and / or the apoptosis inhibitor is a Rho kinase inhibitor; The operation is a treatment that causes damage to the in vitro culture, excluding freezing and thawing operations, The damage involves damage or alteration of various structures necessary for cell survival and normal differentiation, and double-strand breaks in DNA within the nucleus. A method for suppressing embryonic development arrest.

15. The in vitro culture is from the order Primates, Rodents, Lagomorpha, Cetartiodactyla, Perissodactyla, or Carnivora. The method for suppressing embryonic development arrest according to claim 14.

16. The specific period is within one hour when using animal species, strains, and / or frozen eggs that are sensitive to the procedure. The method for suppressing embryonic development arrest according to claim 14 or 15.

17. The in vitro culture is derived from a mammalian strain that is susceptible to the manipulation. The method for suppressing embryonic development arrest according to claim 16.

18. The procedure is a multiple electroporation method, The mammal is a crossbred, inbred, disease model, or xenogenic The method for suppressing embryonic development arrest according to claim 17.

19. A developmentally engineered product including any one or any combination of non-human individuals, organs, tissues, and cells is produced from the in vitro culture treated by the method for suppressing embryonic development arrest according to any one of claims 14 to 18. A method for producing a developmentally engineered product.

20. The in vitro culture treated by the method for suppressing embryonic development arrest according to any one of claims 14 to 18, and / or The developmentally engineered product produced by the method of producing a developmentally engineered product according to claim 19 is transplanted into a recipient other than a human. A transplantation method characterized by:

21. A method for treating a mammal, other than a human, comprising: The in vitro culture treated by the method for suppressing embryonic development arrest according to any one of claims 14 to 18, and / or The developmentally engineered product produced by the method of producing a developmentally engineered product according to claim 19 is transplanted into a recipient other than a human. A method of treatment characterized by:

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