Composition for embryo development containing Rad51 activator and method for improving embryo development rate using the same

Abstract

One aspect provides a method for increasing the efficiency of somatic cell nuclear transfer using a substance (RS-1) that increases Rad51 activity, and somatic cell nuclear replacement cells produced thereby. Another aspect provides a method for screening substances that increase Rad51 activity in order to increase the efficiency of somatic cell nuclear replacement. The efficiency of somatic cell nuclear replacement can be increased by using a substance that increases Rad51 activity. [Selected Figure] Figure 3A

Description

[Technical field]

[0001] The present invention relates to a composition for embryo development comprising a Rad51 activator, and a method for improving the rate of embryo development using the same. [Background technology]

[0002] For the purpose of producing patient-immunocompatible stem cells, the most advantageous method is to produce embryonic stem cells via somatic cell nuclear replacement. However, the production efficiency of somatic cell nuclear replacement technology is very low, and the reasons for failure have not been properly investigated. Therefore, the issues that must be resolved for commercialization of somatic cell nuclear replacement stem cells are to determine the cause of the low production efficiency and to increase the production efficiency.

[0003] For the homeostasis of the genome, DNA must be preserved without damage. The most fatal DNA damage is the break in the DNA double helix, and Rad51 is one of the proteins involved in the restoration of the broken DNA double strand. When a DNA double strand break occurs, exonuclease first forms a single strand at the break site, and the single strand is protected by being coated by replication protein A (RPA). Then, the recombination protein Rad51 replaces RPA and forms a filament-shaped complex, searches for homologous chromosomes, finds homologous base sequences, and exchanges DNA strands to complete the homologous recombination repair.

[0004] In addition, RS-1, a chemical that increases the enzymatic activity of Rad51, is known to improve the efficiency of homologous recombination by increasing the DNA binding activity of Rad51. Summary of the Invention [Problem to be solved by the invention]

[0005] One aspect is to provide a composition for embryo development that includes an agent that increases the activity of Rad51.

[0006] Another aspect is to provide a method for increasing the efficiency of embryo development comprising culturing eggs in medium containing an agent that increases Rad51 activity. [Means for solving the problem]

[0007] One aspect provides a composition for increasing the efficiency of embryo formation or development in a fertilized egg, comprising a substance that increases Rad51 activity. Another aspect provides a composition for increasing the efficiency of embryo formation or development, comprising a Rad51 activator.

[0008] As used herein, the term "embryo formation" means that a zygote undergoes cell division to give rise to several cells, which then undergo cell division and differentiation to form or develop into an embryo.

[0009] As used herein, the term "increased efficiency" refers to increased blastocyst development or blastocyst development of somatically replicated eggs. The term "blastocyst" refers to a fertilized egg that has developed to the point where it is divided into an inner cell mass that forms a blastocyst cavity and differentiates into a fetus and a trophectoderm that differentiates into a placenta after a compacted morula has formed during the process of repeated cleavage of the fertilized egg. Thus, the increased efficiency refers to an increased efficiency of somatic cell nuclear transfer compared to somatic cell nuclear transfer performed in the absence of a preparation that reduces H3K9me3 methylation, specifically, an increased efficiency of embryo development of somatically replicated eggs, an increased development of somatic cell nuclear transfer embryos to the blastocyst stage, an increased development to the blastocyst stage, an increased efficiency of blastocyst production, an increased efficiency of obtaining blastocysts, or an increased rate of blastocyst development and formation.

[0010] The term "Rad51" as used herein refers to a gene expressed in eukaryotes and involved in the repair of DNA double-strand breaks, i.e., a member of the Rad51 protein family as a DNA corrector. The sequence and location of the gene are known in the art (e.g., NCBI Gene ID: 5888).

[0011] The substance that increases the activity of Rad51 may be, for example, a commercially available product named RS-1 (RAD51-stimulatory compound-1), or may be a substance that binds to Rad51 selected by a method known in the art, such as high throughput screening (HTS), or may include 3-[(benzylamino)sulfonyl]-4-bromo-N-(4-bromophenyl)benzamide, 4-bromo-N-(4-bromophenyl)-3-[[(phenylmethyl)amino]sulfonyl]-benzamide, or a compound of the following formula 1 or a derivative thereof:

[0012] [ka] The substance that increases the activity of Rad51 enhances the activity of Rad51 protein by maintaining the binding safety of Rad51 protein, which has the function of correcting damaged DNA, to ssRNA (single strand DNA) or dsDNA (double strand DNA), and therefore can have the function of sustaining the DNA correction effect.

[0013] In one embodiment, the medium composition may be a basal medium containing a substance that increases Rad51 activity at a concentration of 0.1 μM to 50 μM. Specifically, the basal medium may be a basal medium used for culturing mammalian oocytes. The basal medium may include any of the common media known to those skilled in the art, including at least one selected from the group consisting of inorganic salts, carbon sources, amino acids, bovine serum albumin, and cofactors, depending on the species of mammals. The inorganic salts may include NaCl, KCl, NaHCO3, etc., the carbon sources may include glucose, sodium, pyruvate, calcium lactate, etc., the amino acids may include essential amino acids such as glutamine and non-essential amino acids, and the cofactors may include other trace elements and buffer solutions. The medium may be selected from the group consisting of, for example, Minimal Essential Medium (MEM), Dulbecco Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Keratinocyte Serum Free Medium (K-SFM), Iscove's Modified Dulbecco's Medium (IMDM), F12, and DMEM / F12. The medium may also contain a neutral buffer (e.g., phosphate and / or high-concentration bicarbonate) and protein nutrients (e.g., serum, e.g., FBS, FCS (fetal calf serum), horse serum, serum substitute, albumin, or essential and non-essential amino acids, e.g., glutamine, L-glutamine) in an isotonic solution. Additionally, it may contain lipids (fatty acids, cholesterol, HDL or LDL extracts of serum) and other components found in most storage media of its type (e.g., transferrin, nucleosides or nucleotides, pyruvate, a sugar source in any ionized form or salt, e.g., glucose, a glucocorticoid, e.g., hydrocortisone, and / or a reducing agent, e.g., β-mercaptoethanol).The basal medium may also further contain antibiotics.The substance that increases Rad51 activity may be included in the basal medium at a concentration of 0.1 μM to 50 μM, 0.1 μM to 40 μM, 0.1 μM to 30 μM, 1 μM to 50 μM, 1 μM to 30 μM, 5 μM to 30 μM, or 5 μM to 25 μM. If the concentration of the substance that increases Rad51 activity included in the basal medium is less than the above range, it is not possible to effectively remove reactive oxygen, which inhibits the blastocyst development efficiency of eggs, making it difficult to obtain eggs of good quality. If the concentration exceeds the above range, the substance that increases Rad51 activity acts on eggs for a long period of time, hindering the maturation of eggs.

[0014] In one embodiment, the composition may further comprise an agent that reduces H3K9me3 methylation. The agent that reduces methylation may be an agent that increases expression of a member of the KDM4 family of histone demethylases. For example, the agent may increase expression or activity of KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD2D), or a combination thereof.

[0015] Another aspect provides a method for increasing the efficiency of oocyte embryo development, comprising culturing oocytes in a medium containing a substance that enhances Rad51 activation, the specific contents of which are as described above.

[0016] In one embodiment, the method may include contacting an ovum with a preparation that reduces H3K9me3 methylation. The specific contents of the preparation that reduces H3K9me3 methylation are as described above. The ovum may be thawed after being frozen.

[0017] The method may include culturing oocytes for 1 to 10 days in a medium containing a substance that increases Rad51 activation. Specifically, the oocytes may be cultured for 1 to 10 days, 1 to 8 days, 1 to 7 days, 2 to 8 days, 2 to 6 days, or 3 to 6 days. If the culture period is shorter than the above range, there is a problem that the embryos are not sufficiently developed into blastocysts, and if the culture period exceeds the above range, there is a problem that the oocytes are overly matured, making it difficult to obtain blastocysts with improved quality.

[0018] The method may further comprise developing the embryo obtained from the culturing step into an individual, which may be a morula, a blastula, and / or an oocyte.

[0019] Another aspect provides an embryo, blastocyst and / or embryonic stem cell produced by the method. Yet another aspect provides a transplant composition comprising as an active ingredient the embryo and / or blastocyst produced by the method.

[0020] Another aspect provides a method for increasing the efficiency of somatic cell nuclear transfer using a substance that increases Rad51 activity. The efficiency can be the success rate of somatic cell nuclear transfer or the rate of embryonic development of cells generated by the somatic cell nuclear transfer. The rate of embryonic development can refer to the development of an embryo through the 2-cell, 4-cell, and 8-cell stages to a blastocyst.

[0021] The present inventors confirmed that the expression level of Rad51 is very low in the process of cell replication produced by somatic cell nuclear transfer compared to the process of in vitro fertilization, and confirmed that a Rad51 activity enhancer dramatically improves the efficiency of somatic cell replication. Therefore, Rad51 activators are useful for cell production by somatic cell nuclear transfer, and by selecting a substance that enhances Rad51 activity, the efficiency of somatic cell nuclear transfer can be significantly increased.

[0022] The method may include the steps of providing a somatic cell nucleus and an egg from which the nucleus has been removed, culturing the somatic cell nucleus and the egg from which the nucleus has been removed with a substance that increases Rad51 activity, and obtaining a somatic-cell nuclear transfer (SCNT) cell.

[0023] The method may include the steps of removing the oocyte nucleus, transferring one or more somatic cell nuclei (donor nuclei), activating the reconstructed nuclear-transferred oocyte (germ), and further culturing it into a blastocyst. Such steps for somatic cell nuclear transfer (SCNT) can be performed by a person of ordinary skill in the art with appropriate modifications according to methods published in the literature (Nature 419, 583-587, 10 October 2002) and the like.

[0024] The method may include the step of adding one or more somatic cells, i.e., one or more nuclei of donor cells, to the oocyte, including a donor nucleus via direct injection or electrical stimulation, and fusing the nucleus and cell.

[0025] "Somatic cell nuclear replacement" or "somatic cell nuclear transfer" refers to the technique of transplanting a nucleus from a donor cell into a recipient cell from which the nucleus has been removed, resulting in the development of a cell that is genetically identical to the donor cell.

[0026] The enucleated eggs may be cumulus-oocyte complexes collected from the follicles of an individual, or may be commercially obtained. The individual may be a mammal, including a human. For example, the mammal may include a mouse, a pig, a sheep, a dog, a cow, a horse, a goat, etc. The enucleated eggs may be frozen and / or cryopreserved. The freezing method may be a slow-freezing method or a vitrification method, which is an ultra-rapid freezing method.

[0027] A method according to one embodiment may include culturing the somatic cell nucleus and the enucleated egg in vitro to form a blastocyst.

[0028] The substance that increases the activity of Rad51 may also be added to a medium for culturing the nuclei of somatic cells and oocytes from which the nucleus has been removed, and may be present in the medium, for example, before or after generation of SCNT cells, at the 2-cell stage, 4-cell stage, 8-cell stage, or during blastocyst formation. The concentration of the substance that increases the activity of Rad51 may be appropriately adjusted by a skilled artisan depending on the degree of Rad51 activation or cytotoxicity of the substance, and may be present in the medium at a concentration of, for example, 0.1 μM to 50 μM, 0.1 μM to 40 μM, 0.1 μM to 30 μM, 1 μM to 50 μM, 1 μM to 30 μM, 5 μM to 30 μM, or 5 μM to 25 μM.

[0029] According to one embodiment, the method may include contacting with an agent that reduces H3K9me3 methylation. The agent that reduces H3K9me3 methylation may be an agent that increases expression of a member of the KDM4 family of histone demethylases. The agent may, for example, increase expression or activity of KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD2D), or a combination thereof. The contacting may be performed on a nuclear transfer embryo after fusing a somatic cell nucleus and an enucleated egg. In particular, the embryo may be an embryo before somatic cell nuclear transfer oocyte gene activation begins. For example, the embryos can be contacted with one or more KDM4 family histone demethylases for 5 hours post activation (5 hpa), or between 10 and 12 hpa (i.e., 1-cell stage), or at about 20 hpa (i.e., early 2-cell stage), or between 20 and 28 hpa (i.e., 2-cell stage).

[0030] The contacting or injecting step may involve contacting or injecting one or more histone demethylases of the KDM4 family into a donor cell, e.g., a terminally differentiated somatic cell nucleus or cytoplasm, prior to injecting the donor cell nucleus into an oocyte whose enucleation has been removed. The contacting or injecting step may involve contacting the donor somatic cell for 1 hour or more, or 2 hours or more, and the contacting may be performed 1 day (24 hours), 2 days or more, 3 days or more, or more than 3 days prior to the removal of the oocyte whose enucleation has been removed from the donor somatic cell.

[0031] Therefore, by contacting the somatic cell nucleus and the egg from which the nucleus has been removed with a preparation that reduces H3K9me3 methylation, the activity of histone methylases that inhibited replication in the SCNT embryo is reduced, and the expression of genes related to embryo development is enhanced, thereby improving the development and development efficiency of the blastocyst. For example, the development efficiency can be increased by increasing the development percentage (%) of the SCNT embryo that develops to the 2-cell, 4-cell, and 8-cell or blastocyst stages. That is, the development efficiency can be increased before the SCNT embryo is transferred to the 8-cell or blastocyst stage. In one embodiment, changes in the embryo state and the number of blastocysts were confirmed depending on the presence or absence of a substance that increases Rad51 activity during the SCNT process. As a result, it was confirmed that the number of embryos blocked at the 2-cell stage was reduced and the embryo development rate was significantly increased by treatment with a substance that increases Rad51 activity (FIG. 3A). Therefore, a substance that increases the Rad51 activity overcomes the 2-cell stage developmental arrest phenomenon and maintains continuous embryo development, thereby increasing the efficiency of blastocyst development.

[0032] In one embodiment, the method increases the efficiency of nuclear transfer by about 5% or more, about 10% or more, about 13% or more, about 15% or more, about 30% or more, about 50% or more, 50-80%, or more than 80%, compared to SCNT performed in the absence of an agent that increases Rad51 activity, i.e., increases the efficiency of pre-implantation development of SCNT embryos, increases the development of embryos to the blastocyst stage, or increases the development of embryos to the expanded blastocyst stage by about 5%, about 7%, about 10%, about 12% or more, or increases the development of embryos to the expanded blastocyst stage by about 5%. In yet another embodiment, the method increases successful development to the blastocyst stage by 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, compared to SCNT performed in the absence of an agent that increases Rad51 activity. The increase in somatic cell nuclear transfer efficiency means an increase in the production or acquisition of blastocysts. The increase in the production or acquisition of blastocysts may be an increase in the production or acquisition of blastocysts of about 110% or more, about 120% or more, about 130% or more, about 140% or more, about 150% or more, compared to somatic cell nuclear transfer performed in the absence of a substance that increases Rad51 activity.

[0033] As described above, in the method according to one aspect, SCNT oocytes are cultured in a medium containing a substance that enhances Rad51 activity, which prevents cell damage to SCNT oocytes and improves the quality of the oocytes. In addition, the Rad51 activator reduces cell death in SCNT oocytes that have been frozen and thawed, which improves the efficiency of blastocyst formation and production. In addition, the implantation rate of SCNT oocytes is improved, which allows the production of endangered animals through in vitro fertilization, and embryonic stem cell induction is enhanced, which allows the efficient production of embryonic stem cell lines.

[0034] Another aspect provides a somatic cell nuclear transfer embryo, blastocyst, embryonic stem cell, or cloned germ cell produced by the method. The embryo may be genetically modified, e.g., modified in one or more transgenes in the genetic material of the donor nucleus prior to the somatic cell nuclear transfer step (i.e., prior to harvesting the donor nucleus and fusing it with the cytoplasm of the recipient oocyte). In one embodiment, the embryo may comprise nuclear DNA from a donor somatic cell, cytoplasm from a recipient oocyte, and mitochondrial DNA from a third donor individual. The embryonic stem cell may be formed by isolating cells from the inner cell mass of a blastocyst produced by the method and culturing the undifferentiated inner cell mass-derived cells. The embryonic stem cell may also be a pluripotent stem cell or a totipotent stem cell.

[0035] Another aspect provides a method for screening for a substance that increases Rad51 activity and increases somatic cell replication efficiency, comprising the steps of preparing a cell nucleus and an egg from which the nucleus has been removed, culturing the somatic cell nucleus and the egg from which the nucleus has been removed with a candidate substance, and evaluating the Rad51 activity of somatic cell nucleus-replaced cells generated after the culture.

[0036] The cells derived from the SCNT embryo or blastocyst, such as embryonic stem cells, can also be used to test whether a preparation affects differentiation or cell proliferation. For example, the ability of the cells to differentiate or proliferate is evaluated from the presence or absence of the preparation, which can be used to screen for preparations that affect the cells derived from the SCNT embryo or blastocyst. The test compound can be any compound of interest, including chemical compounds, small molecules, polypeptides, or other biological agents (such as antibodies or cytokines).

[0037] In one embodiment of the method, the step of evaluating Rad51 activity is to confirm the expression level of Rad51, and can be performed using techniques well known in the art, such as RT-PCR or immunostaining. The step of evaluating Rad51 activity can also be performed together with the step of evaluating somatic cell replication efficiency. If the number of SCNT oocytes, 2-cell stage cells, 4-cell stage cells, or blastocyst stage cells in the culture changes after addition of the candidate substance compared to before addition, the candidate substance can be determined to be a substance that increases the activity of Rad51 and further increases the efficiency of somatic cell replication by somatic cell nuclear transfer.

[0038] Another aspect provides a method for producing a SCNT fertilized egg, comprising the steps of preparing a somatic cell nucleus and an egg from which the nucleus has been removed, culturing the somatic cell nucleus and the egg from which the nucleus has been removed in a medium containing a substance that enhances Rad51 activity to obtain a SCNT cell, and fertilizing the SCNT cell and liquid semen in vitro. The details of the method for culturing the somatic cell nucleus and the egg from which the nucleus has been removed in a medium containing a Rad51 activator are as described above. The SCNT cell may be a SCNT egg, specifically, a 2-cell stage, 4-cell stage, or 8-cell stage embryo, or a cell from which the embryo has developed to reach the blastocyst stage.

[0039] According to one embodiment, the method includes a step of in vitro fertilization of the SCNT cells and liquid semen. The semen may be collected from the vas deferens of an individual. The individual may be a mammal, including a human. For example, the mammal may include a mouse, a pig, a sheep, a dog, a cow, a horse, a goat, and the like. The SCNT cells and liquid semen may be in vitro fertilized in a medium composition for in vitro fertilization for 1 to 7 days. If the in vitro fertilization period is shorter than the above range, there is a problem that fertilization does not occur, and if it exceeds the above range, there is a problem that the embryo degenerates. The above step may further include a step of contacting or injecting a substance that enhances Rad51 activity. Specific details regarding the substance that enhances Rad51 activity are as described above. The substance that increases the activity of Rad51 can be contacted with or injected into the SCNT fertilized egg before pronucleus formation (within 18 hours after sperm injection) after the SCNT cell is injected with sperm, specifically before the 2-cell stage after the sperm injection. Thus, the SCNT fertilized egg can increase production efficiency because the SCNT cell (embryo) can progress efficiently without developmental arrest and successfully develop into a blastocyst through the 2-cell stage, 4-cell stage, and 8-cell stage without developmental defects or loss of viability. Effect of the Invention

[0040] The present invention relates to a composition for embryo development containing a Rad51 activator, and a method for improving the rate of embryo development using the same, in which the cells can be efficiently developed into blastocysts without cell damage and / or developmental arrest, and can be used to produce somatic cell cloned fertilized eggs using in vitro fertilization procedures. [Brief description of the drawings]

[0041] [Figure 1A] The results of RT-PCR analysis of the level of Rad51 expression during IVF, SCNT using fresh oocytes, and SCNT involving injection of Kdm4a mRNA into oocytes are shown. [Figure 1B]The results of RT-PCR analysis of the expression level of Rad51 during IVF, SCNT using frozen oocytes, and SCNT using fresh oocytes are shown. [Diagram 2] The results of immunostaining analysis of the Rad51 expression level in IVF, SCNT using frozen oocytes, and SCNT using fresh oocytes are shown. [Figure 3A] 1 shows photographs showing the change in the number of embryos and blastocysts in a state of developmental arrest at the 2-cell stage during the SCNT process in the presence or absence of a Rad51 activator. [Figure 3B] These are photographs confirming the correlation between autophagy and mitochondria in IVF oocytes, SCNT oocytes, and SCNT+RS-1 oocytes. [Figure 4] These are photographs confirming mitochondrial activity in IVF oocytes, SCNT oocytes, and SCNT+Kdm4a oocytes. [Figure 5A] These are photographs confirming cell damage in IVF eggs, SCNT eggs, and SCNT+RS-1 eggs. [Figure 5B] These are photographs confirming the expression of DNA damage biomarkers in IVF oocytes, SCNT oocytes, and SCNT+RS-1 oocytes. [Figure 5C] These are photographs confirming DNA damage by cell cycle in IVF oocytes, SCNT oocytes, and SCNT+RS-1 oocytes. [Figure 6] This is a graph confirming the degree of DNA breakage in IVF oocytes, SCNT oocytes, and SCNT+RS-1 oocytes. [Figure 7] This is a graph confirming the effect of RS-1 treatment on SCNT oocytes through the increase or decrease in gene expression. [Figure 8] These are photographs showing the mechanism patterns of SCNT by RS-1 treatment and Kdm4a mRNA treatment, confirmed by H3K9me3 staining. [Figure 9A] This is a table showing the replication production rate of SCNT oocytes depending on the treatment with RS-1. [Figure 9B] 13 is a graph confirming the embryonic stem cell induction rate of SCNT oocytes depending on RS-1 treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] An embodiment of the present invention will be described below. (1) A composition for increasing the efficiency of embryo development or embryo formation, comprising a substance that increases Rad51 activity. (2) The composition according to (1), wherein the substance that increases the activity of Rad51 is a compound represented by the following chemical formula 1 or a derivative thereof: [ka] (3) The composition described in (1), further comprising a preparation that reduces H3K9me3 methylation. (4) The composition described in (3), wherein the formulation that reduces H3K9me3 methylation is a formulation that increases expression of a member of the KDM4 family of histone demethylases. (5) The composition described in (4), wherein the formulation increases the expression or activity of KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD2D), or a combination thereof. (6) The method according to (1), wherein the concentration of the substance that increases the activity of Rad51 is 5 μM to 15 μM. (7) The composition described in (1), wherein the substance that increases Rad51 activity is present at the 4-cell stage of an embryo. (8) The composition described in (1), wherein the embryo development is the development of an embryo into a blastocyst or the formation of a blastocyst. (9) The composition described in (1), wherein the embryo is formed in vitro through artificial fertilization. (10) The composition described in (9), wherein a substance that increases the activity of Rad51 is present after sperm is injected into an egg in the artificial insemination and before pronuclear formation. (11) The composition described in (1), wherein the embryo is formed by transplanting the nucleus of a somatic cell into an egg from which the nucleus has been removed. (12) A method for increasing the efficiency of embryo development or the formation of embryos, comprising culturing eggs in a medium containing a substance that increases Rad51 activity. (13) The method according to (12), comprising the step of contacting the egg with a preparation that reduces H3K9me3 methylation. (14) The method according to (12), wherein the eggs have been frozen and then thawed. (15) The method according to (12), wherein the embryo obtained by the culture has induced damage in DNA replication. (16) The method according to (12), wherein the damage is induced after the 2-cell stage and before the 4-cell stage. (17) The method according to (12), wherein the substance that increases the activity of Rad51 reduces the developmental arrest at the 2-cell stage of the embryo obtained by the culture. (18) The method according to (12), further comprising a step of developing the embryo obtained in the culturing step into an individual. (19) An embryo produced by the method according to (12). (20) A blastocyst produced by the method according to (12). (21) An embryonic stem cell produced by the method according to (12). The present invention will be described in more detail below with reference to examples. However, these examples are merely for illustrative purposes and the scope of the present invention is not limited to these examples.

[0043] Example 1. Somatic cell nuclear transfer (SCNT) 5IU PMSG hormone and 5IU hCG hormone were administered to 8-10 week old female BDF / 1 mice. 14 hours after hCG administration, cumulus cells were isolated from the superovulated mice using hyaluronidase, and oocytes were collected. The isolated cumulus cells were stored in a refrigerator to be used as donor cells for somatic cell replication, and the isolated oocytes were stored in a KSOM culture medium incubator at 37°C until the start of the experiment. Then, for the somatic cell replication experiment, the nuclei were removed from the matured oocytes, and the previously isolated cumulus cells were directly injected into the donor cells. The cumulus cells injected into the donor cells were artificially activated in a culture medium containing RS-1 (Rad51-stimulatimulatory compound 1) for 6 hours. Then, the mice were cultured for 22 hours in a culture medium containing the RS-1 reagent, and after 22 hours, they were cultured in KSOM culture medium for 72 to 96 hours.

[0044] Example 2. Preparation of Kdm4a mRNA and injection of Kdm4a mRNA into somatic cell-replicated oocytes 2-1.Preparation of Kdm4a mRNA Full-length mouse Kdm4a / Jhdm3a cDNA was cloned into pcDNA3.1 plasmid containing poly(A)83 at the 3' end of the cloning site using the In-Fusion Kit (Clonetech #638909). From the template plasmid linearized by in vitro transcription, mRNA was synthesized using mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies #AM1345). The synthesized mRNA was dissolved in nuclease-free water. The concentration of mRNA was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). An aliquot of mRNA was stored at -80°C until use.

[0045] 2-2. Injection of Kdm4a mRNA into SCNT oocytes 2μg / μl of Kdm4a mRNA prepared in Example 2-1 was injected into the replicated oocytes treated with RS-1 for 6 hours and the replicated oocytes not treated with RS-1, together with 10pl of the control group, in the activation culture medium using a piezo-driven micromanipulator. After the injection, the replicated oocytes were cultured for 22 hours in the KSOM culture medium containing RS-1 and in the culture medium not containing RS-1, respectively, and then cultured for 72 to 96 hours in the culture medium not containing RS-1, and the blastocyst efficiency was observed.

[0046] Example 3. Comparative analysis of Rad51 expression levels in in vitro fertilized eggs and somatic cell nuclear transfer eggs The expression levels of Rad51, which regulates the homologous recombination mechanism, in in vitro fertilized (IVF) eggs, somatic cell nuclear transfer eggs (SCNT, F / T SCNT) in Example 1, and cloned eggs (SCNT+Kdm4a) injected with Kdm4a mRNA during the somatic cell nuclear transfer process in Example 2 were comparatively analyzed by qRT-PCR. Specifically, embryo mRNA was isolated using Dynabeads mRNA DIRECT kit (Dynal Asa, Oslo, Norway). Approximately 20 embryos at the pronuclear stage (PN), 2-cell stage, and 4-cell stage were washed with lysis / binding buffer and then incubated with Dynabeads oligo dT 25 and allowed to bind at room temperature. After the mRNA was bound to the beads, they were washed twice with Buffer-A and then with Buffer-B. Then, Tris-HCl solution was added and the mRNA was isolated from the beads at 73°C. cDNA was synthesized from the isolated mRNA using an oligo dT primer. Using the synthesized cDNA as a template, the expression of mouse Rad51 was analyzed by qRT-PCR. The primer sequences used were as follows: Forward primer: 5'-AGCTTTCAGCCAGGCAAAT-3' Reverse primer: 5'-GCTTCAGCTTCAGGAAGACA-3' A comparative analysis of Rad51 expression in embryos at the pronuclear stage, 2-cell stage, and 4-cell stage showed that Rad51 was highly abundantly expressed at the 1-cell stage of IVF, tended to decrease significantly at the 2-cell stage, and showed an increase in Rad51 expression at the 4-cell stage, similar to that at the 1-cell stage. On the other hand, in SCNT, the expression pattern was similar to that of IVF up to the 1-cell and 2-cell stages, but at the 4-cell stage, no increase in expression similar to that at the 1-cell stage of SCNT was observed compared to IVF. However, in SCNT+Kdm4a, an expression pattern similar to that of IVF was observed (Fig. 1A).

[0047] We confirmed that Rad51 expression in 4-cell stage embryos generated by SCNT using fresh and frozen oocytes was significantly lower than that in 4-cell stage embryos generated by IVF (FIG. 1B).

[0048] Next, we reanalyzed the results of the significantly lower Rad51 RNA expression in 4-cell oocytes formed by SCNT compared to 4-cell oocytes formed by IVF using immunostaining. Specifically, after 30 hours of SCNT activation, at the 2-cell stage, the oocytes were washed with PBS containing 0.1% BSA and treated with 4% paraformaldehyde for 30 minutes at room temperature. After 24 hours of treatment with PBS / 0.1% BSA containing 0.1% Triton X, the oocytes were incubated with Rad51, γH2AX, Mitotracker, and LC3B antibodies for 2 hours at room temperature. The oocytes were then washed three times for 10 minutes with PBS containing 0.1% BSA, and treated with goat anti-mouse and donkey anti-rabbit antibodies for 1 hour each. After washing three times for 10 min with PBS / 0.1% BSA, nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and the stained oocytes were fixed on slides and analyzed by confocal fluorescence microscopy.

[0049] As a result, we found that up to the 2-cell stage, there was no difference in the amount of Rad51 protein among IVF, SCNT, and SCNT using frozen eggs, but at the 4-cell stage formed by somatic cell nuclear replacement, the amount of Rad51 protein was significantly lower (Figure 2).

[0050] Example 4. Confirmation of improvement in somatic cell replication efficiency by treatment with Rad51 activity-increasing compound and biomarker confirmation 4-1. Confirmation of improvement in somatic cell replication efficiency Based on the results of Example 3, we analyzed whether the efficiency of somatic cell replication is improved after treatment with RS-1, a substance that increases Rad51 activity, during the somatic cell replication process. First, to confirm whether RS-1 induces toxicity in oocyte development, 0, 1, 5, and 10 μM of RS-1 were treated with the in vitro fertilized (IVF) oocytes and the somatic cell nuclear transfer oocytes (SCNT, F / T SCNT) in Example 1 to select the appropriate concentration.

[0051] As a result, we confirmed that at a concentration of 10 μM, the embryo development rate increased significantly (Table 1) (control group: 31%, RS-1: 76%). As expected, RS-1 treatment also reduced the number of embryos in a 2-cell stage developmentally blocked state (control group: 39%, RS-1: 15%), and we confirmed that the embryo development rate also increased significantly (control group: 17%, RS-1: 59%) (Figure 3A).

[0052] [Table 1] 4-2. Confirmation of biomarker expression and correlation The correlation between autophagy and mitochondria was confirmed through staining with LC3B, an autophagy marker, and Mito Tracker, a mitochondrial marker, at the 1-cell, 2-cell, and 4-cell stages of in vitro fertilized (IVF) oocytes, somatic cell nuclear transfer oocytes (SCNT, F / T SCNT) in Example 1, and somatic cell nuclear transfer oocytes treated with RS-1 (SCNT+RS-1) in Example 4-1. Specifically, somatic cell replicated oocytes at the 1-cell, 2-cell, and 4-cell stages derived from SCNT were stained with Mito Tracker Orange CMTMRos (Molecular Probes) to evaluate mitochondrial distribution. Mito Tracker was used in M16 culture medium supplemented with 0.3% BSA at a concentration of 300 nM for 30 minutes at 37° C. under dark conditions. After washing, the oocytes were fixed and immunofluorescently stained with LC3B antibody, and then the nuclei were stained with DAPI.

[0053] As a result, at the 1-cell stage, only in the SCNT group, the distribution of autophagy and mitochondria was condensed in the cytoplasm, and a unique point was found in which they were expressed as small dots, and in the SCNT group treated with RS-1, an expression pattern similar to that of the IVF group was confirmed. At the 2-cell stage, the expression of autophagy and mitochondria was distributed throughout the nucleus and cytoplasm in the IVF group, but in the SCNT group and the SCNT + RS-1 group, autophagy and mitochondria were concentrated in the nucleus. In particular, in the SCNT group, which underwent developmental arrest at the 2-cell stage, a unique point was confirmed in which expression was not observed in the nucleus, but was expressed only in the cytoplasm. At the 4-cell stage, the expression pattern was similar in all groups (Figure 3B).

[0054] Example 5. Analysis of mitochondrial activity in in vitro fertilized eggs and somatic cell nuclear transfer eggs In order to analyze mitochondrial activity and potential in in vitro fertilized (IVF) eggs, somatic cell nuclear transfer eggs (SCNT, F / T SCNT) in Example 1, and cloned eggs injected with Kdm4a mRNA during the somatic cell nuclear transfer process (SCNT+Kdm4a) in Example 2, each egg was stained with JC-1. Specifically, to measure mitochondrial activity, 1-cell stage, 2-cell stage, and 4-cell stage SCNT-derived somatic cell cloned eggs were added to the culture medium at a concentration of 1 μg / ml using JC-1 (Thermo Fisher Scientific, Waltham, MA, USA) and cultured in the dark for 20 minutes. Then, the nuclei were stained with Hoechst (Sigma).

[0055] As a result, in the IVF group, it was confirmed that mitochondria were abundantly expressed in the cytoplasm and around the nucleus at the 2-cell stage. On the other hand, in the SCNT group and the SCNT+Kdm4a group, it was confirmed that mitochondria were partially condensed in the cytoplasm and expressed unstably. Furthermore, it was confirmed that mitochondria were relatively expressed only on the cytoplasmic surface at the 4-cell stage in the SCNT group and the SCNT+Kdm4a group. Therefore, it can be seen that the mitochondrial expression patterns in the IVF group and the SCNT group are completely different (Figure 4).

[0056] Example 6. Confirmation of cell damage and DNA damage caused by RS-1 treatment 6-1. Confirmation of cell damage In order to confirm the cellular damage of SCNT eggs by RS-1 treatment, the generation of reactive oxygen species was confirmed at the 2-cell stage in the in vitro fertilized (IVF) eggs, the SCNT eggs (SCNT, F / T SCNT) in Example 1, and the SCNT eggs (SCNT+RS-1) treated with RS-1 in Example 4-1. Specifically, the IVF group, the SCNT+RS-1 group and the SCNT group at the 2-cell stage were cultured at 37°C in a dark condition for 30 minutes in a culture medium containing 5 μM of CellROX oxidative stress reagent. After that, the eggs were washed with D-PBS containing 0.1% PVA, and the nuclei were stained with Hoechst (Sigma).

[0057] As a result, it was confirmed that the IVF group showed a tendency to show an overall decrease in reactive oxygen species, while the SCNT+RS-1 and SCNT groups showed an increase in reactive oxygen species in their nuclei (Figure 5A).

[0058] 6-2. Confirmation of DNA damage In order to confirm the DNA damage of SCNT eggs treated with RS-1, the expression of DNA damage biomarker (rH2AX) was confirmed in in vitro fertilized (IVF) eggs, SCNT eggs (SCNT, F / T SCNT) in Example 1, and SCNT eggs (SCNT+RS-1) treated with RS-1 in Example 4-1. Specifically, the IVF group, SCNT group, and SCNT+RS-1 group were fixed with 4% paraformaldehyde at the 1-cell stage, 2-cell stage, and 4-cell stage, and then cultured with rH2AX (Abcam, ab22551) and Rad51 (Abcam, ab63801) for 2 hours, and then cultured with goat anti-rabbit antibody diluted at a concentration similar to that of goat anti-mouse antibody diluted 1:200 in PBS / 0.1% BSA for 1 hour. Then, nuclei were stained with DAPI.

[0059] As a result, it was confirmed that the expression of rH2AX and Rad51 was very negligible in the 1-cell stage of the IVF group. On the other hand, it was confirmed that Rad51 and rH2AX were strongly expressed in the nucleus of the SCNT group, and DNA damage was significantly reduced in the SCNT+RS-1 group. It was also confirmed that DNA damage was strongly expressed in the nucleus of the SCNT+RS-1 2-cell stage developmental arrest group compared to the other groups. It was confirmed that Rad51 and rH2AX were strongly expressed in the 4-cell stage nucleus of the IVF group, and the expression of Rad51 and rH2AXIVF was similar in the 4-cell stage nucleus of the SCNT+RS-1 group. On the other hand, it was confirmed that Rad51 and rH2AX were very negligible in the 4-cell stage nucleus of the SCNT group. In other words, when RS-1 is treated during the somatic cell nuclear transfer process, DNA damage is significantly increased during the process of replication from the 2-cell stage to the 4-cell stage (Figures 5B and 5C).

[0060] Example 7. Confirmation of DNA breaks A single cell electrophoresis (comet assay) was performed to confirm DNA breakage in in vitro fertilized (IVF) eggs, somatic cell nuclear transfer eggs (SCNT, F / T SCNT) in Example 1, and SCNT eggs treated with RS-1. Specifically, to confirm DNA damage, each sample was suspended in 1% agarose at 37°C in a 1 ml tube removed from the culture medium, and then fixed on a pre-coated slide (Trevigen). The slide was incubated at 4°C for 4 hours, and then immersed in Trevigen's 4°C solution for 4 hours. The slide was removed from the dissolution solution, immersed in 1X TAE buffer at 4°C for 30 minutes, and electrophoresed for 30 to 40 minutes. The slide was immersed in 1M ammonium acetate at room temperature for 30 minutes, and then fixed at 75°C. After fixing at room temperature for 30 minutes, the slide was incubated at 42°C for 20 minutes until the agarose was completely dried. Then, the sections were stained with 1X SYBR green I staining solution for 5 minutes.

[0061] As a result, it was confirmed that the 3' end was significantly longer and DNA breaks occurred more frequently in the SCNT group compared to the IVF (in vitro fertilization) group. However, in the SCNT+RS-1 group, which was treated with RS-1, it was confirmed that the 3' end was significantly shorter and DNA breaks were reduced. It was also confirmed that abnormal 3' ends were generated in the SCNT group, but the generation of abnormal 3' ends was significantly reduced in the SCNT+RS-1 group (Figure 6).

[0062] Example 8. Confirmation of the effect of RS-1 treatment on somatic cell nuclear transfer oocytes In order to confirm the effect of RS-1 treatment on SCNT oocytes, the cloned oocytes at the 2-cell stage in the SCNT cloning process of Example 1 were treated with RS-1 and subjected to RNA sequencing. In vitro fertilized (IVF) oocytes were used as a negative control. Specifically, complementary DNA (cDNA) was amplified using a SMARTer Ultra Low Input RNA cDNA Preparation Kit (Takara, 634890) according to the manufacturer's instructions. The cDNA was fragmented into approximately 200 bp fragments using an M220 sonicator (Covaris). The fragmented cDNAs were end-repaired and adapter-ligated. A sequencing library was prepared using a ScriptSeq v2 kit (Illumina) according to the manufacturer's instructions. Single longitudinal sequencing was performed on a HiSeq2500 (Illumina) and mapped to the mm9 mouse genome using STAR (v2.5.2b, https: / / github.com / alexdobin / STAR). Intercepts per kilobase per million reads (FPKM) were then calculated by Cufflink (v2.2.1) using the basic option.

[0063] As a result, it was confirmed that the expression of genes related to translation, immune response, metabolic processes, and mitochondrial translation was increased, and that the SCNT+RS-1 group showed a similar expression pattern to the IVF group. It was also confirmed that the expression of genes related to cell proliferation was increased and the expression of genes related to cell differentiation was decreased (Figure 7).

[0064] Example 9. Confirmation of somatic cell nuclear transfer efficiency by RS-1 treatment and mRNA injection In order to confirm the replication mechanism of the RS-1-treated somatic cell nuclear transfer oocytes (SCNT+RS-1) in Example 3 and the replicated oocytes (SCNT+Kdm4a) injected with Kdm4a mRNA during the somatic cell nuclear transfer process in Example 2, the genes expressed in each oocyte were confirmed using the same method as in Example 8.

[0065] As a result, it was confirmed that there were 190 genes that were significantly increased in the SCNT+RS-1 group, and 45 genes (out of 1,314) that were significantly increased in the SCNT+Kdm4a group. It was also confirmed that there were 414 genes that were significantly decreased in the SCNT+RS-1 group, and 3 genes (out of 478) that were significantly increased in the SCNT+Kdm4a group.

[0066] We also demonstrated that the SCNT induced SCNT by RS-1 and Kdm4a mRNA treatments were mediated by distinct mechanisms through H3K9me3 staining. Specifically, the IVF group, SCNT group, and SCNT+RS-1 group were fixed with paraformaldehyde at the 1-cell stage, and then incubated with H3K9me3 antibody (Millipore, 07-442) for 2 hours, followed by incubation with goat anti-rabbit antibody diluted 1:200 in PBS / 0.1% BSA for 1 hour. Nuclei were then stained with DAPI.

[0067] In other words, it is clear that somatic cell nuclear transfer by RS-1 treatment and Kdm4a mRNA treatment are mediated by completely separate mechanisms (FIG. 8).

[0068] Example 10. Confirmation of replication rate and stem cell induction rate by RS-1 treatment 10-1. Confirmation of the reproduction rate through the production of cloned mice In general, it has been reported that the production rate of cloned mice is around 1% after normal SCNT cloning. Therefore, we confirmed the implantation rate and clone generation rate of mice depending on the treatment of SCNT oocytes with RS-1. Specifically, the embryos were transplanted into the uterus of surrogate female ICR mice 0.5 days after the 2-cell stage of SCNT and SCNT+RS-1 oocytes was pseudopregnant. On the 19.5th day after the transplantation, the SCNT cloned mice were removed from the uterus of the surrogate mother and raised in the surrogate mother's body odor together with normal ICR mice born on the same day as the ICR mice that raised the cloned mice.

[0069] As a result, it was confirmed that the rate of cloned mice produced in the SCNT+RS-1 group treated with RS-1 increased to 5%. In addition, there were no toxicity issues during the developmental process. Therefore, treatment with RS-1 during the production of SCNT oocytes has the advantage of enabling efficient production of cloned mice without toxicity issues (Figure 9A).

[0070] 10-2. Confirmation of stem cell establishment efficiency To confirm the efficiency of stem cell establishment depending on RS-1 treatment, embryonic stem cells were induced from the blastocysts of the SCNTs prepared in Example 1 and the RS-1-treated SCNTs (SCNT+RS-1) prepared in Example 3. Specifically, the blastocysts of each group were cultured on mouse embryonic fibroblast (MEF cells) feeder cells containing mouse embryonic stem cell culture medium. The medium was supplemented with 20% KSR, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, 100 units / ml penicillin, 100 μg / ml streptomycin (all products are from GIBCO), 1.5X10 3 The culture medium was recombinant mLif (Chemicon) at 1000 units / ml. Established somatic cell-replicating mouse embryonic stem cells were passaged using trypsin-EDTA, treated with alkaline phosphatase, and evaluated by histochemical staining.

[0071] As a result, we were able to confirm that the induction rate of embryonic stem cells was significantly higher in the RS-1-treated group (SCNT+RS-1) compared to the RS-1-untreated group (SCNT) (17% vs. 45%) (Figure 9B).

Claims

1. A composition for increasing the efficiency of embryo development or embryo formation of a somatically replicated egg, comprising a substance that increases Rad51 activity and a preparation that reduces H3K9me3 methylation, The substance reduces the number of embryos blocked at the 2-cell stage; The concentration of the substance that increases the activity of Rad51 is 5 μM to 15 μM; The substance that increases the activity of Rad51 is present at the 4-cell stage of an embryo. The composition.

2. The composition according to claim 1, wherein the substance that increases the activity of Rad51 is a compound represented by the following formula 1 or a derivative thereof: 【Chemistry 1】

3. The composition of claim 1, wherein the formulation that reduces H3K9me3 methylation is a formulation that increases expression of a member of the KDM4 family of histone demethylases.

4. 4. The composition of claim 3, wherein the formulation increases expression or activity of KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD2D), or a combination thereof.

5. The composition of claim 1 , wherein the embryo development is the development of an embryo into a blastocyst or the formation of a blastocyst.

6. The composition of claim 1 , wherein the embryo is formed in vitro via artificial insemination.

7. The composition of claim 6, wherein a substance that increases the activity of Rad51 is present after the artificial insemination of an egg and before pronuclear formation.

8. The composition of claim 1 , wherein the embryo is formed by transferring the nucleus of a somatic cell into an egg from which the nucleus has been removed.

9. 1. A method for increasing the efficiency of embryo development or embryo formation of a somatically replicated egg, comprising culturing the egg in a medium containing a substance that increases Rad51 activity and a formulation that reduces H3K9me3 methylation, The substance reduces the number of embryos that are developmentally arrested at the 2-cell stage; The concentration of the substance that increases the activity of Rad51 is 5 μM to 15 μM; The substance that increases the activity of Rad51 is present at the 4-cell stage of an embryo. The method.

10. The method of claim 9, wherein the eggs have been frozen and then thawed.

11. The method according to claim 9, wherein the embryo obtained by the culture is one in which damage in DNA replication is induced.

12. The method of claim 9, wherein the damage is induced after the 2-cell stage and before the 4-cell stage.

13. The method according to claim 9, wherein the substance that increases the activity of Rad51 reduces the 2-cell stage developmental arrest of the embryo obtained by the culture.

14. 10. The method of claim 9, further comprising the step of allowing the embryo obtained from the culturing step to develop into a non-human individual.

Images