WO2019147026A1 - Composition for embryonic development, comprising melatonin, and method for improving efficiency of embryonic development using same - Google Patents

Abstract

The present invention relates to a composition for embryonic development, the composition comprising melatonin, and a method for improving the embryonic development rate using same. The composition prevents the stopping of the development of the 2-cell stage of an embryo, thereby increasing the efficiency of developing into a blastocyst, and thus a blastocyst that is excellent in quality or quantity may be acquired.

Description

A composition for embryo development comprising melatonin and a method for improving the efficiency of embryo development using the composition

Melatonin, and a method for improving the efficiency of embryo development using the same.

Somatic cell nuclear transfer (SCNT) -based reprogramming and derivation of embryonic stem cells (ESCs) can produce patient-specific stem cells for regenerative medicine Has been proposed. Recently, three individual study groups have successfully induced several somatic cell nuclear transfer-embryonic stem cell (SCNT-ESC) lines using high quality human oocytes and fibroblasts from various sources. As much of the developmental arrest of human SCNT embryos has been partially overcome by the modulation of histone methylation, it has become a technology applicable to current cell therapy. In addition, the establishment of human leukocyte antigen (HLA) -modified homozygous pluripotent stem cells (PSCs) may increase the potential efficiency of SCNT-ESCs, but many Lt; RTI ID = 0.0 > human oocytes. ≪ / RTI >

Recently, oocyte vitrification vitrification technology has been used as a practical technique for widely used low-temperature preservation methods as well as human assisted reproductive technology. In fact, it has been reported that cell growth and developmental cell breakdown of offspring born in vitrified (cold-frozen) oocytes did not increase when compared to in vitro fertilization (IVF). This technique provides valuable opportunities to maintain the fertility of infertile women and to cure the risks of fertility due to fertile women and cancer treatment at risk of developing senescence-induced fertility. In addition, the vitrification freezing of human hyperplastic oocytes can provide a continuous oocyte for research, such as SCNT, and its use can also reduce ethical concerns. However, until now, the production of clone using low temperature preserved oocytes and induction of SCNT-ESC system have not been achieved. Even with a survival rate of 90% or more, the clinical outcome of vitrified frozen oocytes was lower than that of fresh oocytes in human reproductive technology programs. This is thought to be due to cytoskeleton damage, changes in spindle structure, microtubule, distribution of cortical granules and hardening of oocytes. In addition, cryopreserved oocytes are particularly vulnerable to oxidative stress due to high levels of lipid and produce large amounts of free radicals that affect the balance between oxidation and reduction reactions and intracellular antioxidant systems.

One aspect is to provide a composition for enhancing embryo development efficiency or embryo formation comprising melatonin.

Another aspect is to provide a method of increasing the efficiency of embryo development or increasing embryo formation, including culturing the oocyte in a medium containing melatonin.

One aspect provides compositions for increasing the formation of embryos, including melatonin. Yet another aspect provides a composition for increasing the efficiency of embryo development, including melatonin. That is, there is provided a composition for increasing the formation of embryos and / or increasing the efficiency of embryo development, including melatonin.

As used herein, the term "embryo formation" means that the zygote becomes a plurality of cells through cell division, and these cells undergo cell division and differentiation to form an embryo or an embryo .

As used herein, the term "increase in efficiency" means an increase in the development of the blastocyst of the oocyte or the development of the blastocyst. The above-mentioned blastocysts can be divided into the inner cell mass to differentiate into the fetus and the trophectoderm that can differentiate into the placenta after the embryo is densified in the process of growing the embryos repeatedly. Means a fertilized egg in a state of being. Thus, the increase in efficiency is due to an increase in the embryo development efficiency of the oocyte, an increase in the development of the embryo to the blastocyst stage compared with the oocyte cultured in the absence of the agent that reduces the H3K9me3 methylation, The increase in the production efficiency of blastocysts, the increase in the efficiency of obtaining blastocysts, or the rate of development of blastocysts.

In one embodiment, the composition may contain melatonin at a concentration of from 0.1 [mu] M to 50 [mu] M in the basal medium. Melatonin is a molecule related to the cycle of biological response, also known as N-acetyl-5-methoxy tryptamine. In mammals, melatonin modulates the cell cycle and exhibits strong resistance to oxidative stress and apoptosis. That is, the method according to one aspect of the present invention reduces the active oxygen and apoptosis of somatic cell nuclear transfer embryos by culturing oocytes in which somatic cells and nuclei are removed in a medium containing melatonin, There is an advantage that an improved blastocyst can be obtained. In addition, somatic cell nuclear transfer embryos can improve the implantation rate of artificially fertilized embryos and promote embryonic stem cell induction. Specifically, in the basic medium, melatonin is added 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, mu M to 20 [mu] M, 10 [mu] M to 25 [mu] M or 5 [mu] M to 15 [mu] M. At this time, when the concentration of melatonin contained in the basic medium is less than the above range, the active oxygen can not be effectively removed, and thus the efficiency of development of the blastocyst of the somatic cell nuclear transfer embryo is deteriorated. As a result, If it exceeds the above range, there is a problem that the melatonin acts on the somatic cell nuclear transfer embryo for a long time and hinders the maturation of the oocyte. Specifically, the basal medium may be a basic medium used for culturing mammalian oocytes. The basic medium differs depending on the species of the mammal, but may include any one or more selected from the group consisting of inorganic salts, carbon sources, amino acids, bovine serum albumin, and coadjuvants, and may include all conventional media known to those skilled in the art. Examples of the inorganic salts include NaCl, KCl, and NaHCO _3. Examples of the carbon source include glucose, sodium, pyruvate, and calcium lactate. Examples of the amino acids include essential amino acids such as glutamine, Other auxiliaries may be other trace elements and buffers. The medium may be, for example, MEM (Minimal Essential Medium), DMEM (Dulbecco modified Eagle Medium), RPMI (Roswell Park Memorial Institute Medium), Keratinocyte Serum Free Medium (K-SFM), Iscove's Modified Dulbecco's Medium And DMEM / F12. ≪ / RTI > The medium may also be supplemented with a mixture of neutral buffer (e.g., phosphate and / or high concentration bicarbonate) and protein nutrients (e.g., serum, such as FBS, fetal calf serum, horse serum, serum replacement, albumin, Amino acids and non-essential amino acids such as glutamine, L-glutamine). Furthermore, it is possible to use lipid (fatty acid, cholesterol, HDL or LDL extract of serum) and other components found in most types of storage medium of this kind (for example, transferrin, nucleoside or nucleotide, pyruvate, For example, glucose, glucocorticoids such as hydrocortisone and / or a reducing agent such as? -Mercaptoethanol). The base medium may further include an antibiotic. The melatonin may be included in the basic medium in 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 . At this time, when the concentration of melatonin contained in the basic medium is less than the above range, the active oxygen can not be effectively removed, and thus the efficiency of development of the blastocyst of the somatic cell nuclear transfer embryo is inhibited. As a result, And if it exceeds the above range, there is a problem that melatonin acts on somatic cell nuclear transfer embryos for a long time to prevent maturation of oocytes.

In other embodiments, the composition may further comprise an agent that reduces H3K9me3 methylation. The agent for decreasing methylation may be one that increases the expression of a member of the KDM4 family of histone demethylating enzymes. For example, the agent may be one that increases the expression or activity of KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD2D), or a combination thereof.

When the embryo is in the 2- celled state, Amd1 , Fam46C , Oxt , Ppt2 , Ctss , Dffa , Eif3f, Hacl1 , Hspbp1 , Mob2 , Mrpl23 , Mrpl33 , Pmvk , Slc4a11 And Lt ; RTI ID = 0.0 > Wbscr22 < / RTI > Also, Atp6v0c, Cd52 , Dapk2 , Ddit4l , Duoxa2 , Nfkbia , Ptgds , Rdh12 And Rnd3 may be decreased.

When the embryo develops into a blastocyst , expression of one or more genes selected from the group consisting of Amd1 , Fam46C , Oxt , Ppt2 , Gulo, and Txnip may be increased. Also, Adh1, Car2, Gsta3, Gstm2 , Mb, Phlda2, S100a1, Akr1c13, Amd2, Clu, Hist1h3a, Hspb1, Il1rn, Nat8, Efemp1, Glipr1, Lgals4, Plac1, Snora15, Snora 21, Snora 34, Anxa1, Clec2f, Hrsp12 , and Plat may be decreased in expression of one or more genes. The embryo may be an embryo formed through artificial fertilization in vitro or the embryo may be formed by implanting nuclei of somatic cells into an oocyte removed from the nucleus. The embryo may be formed through artificial fertilization in vitro or the embryo may be formed by implanting the nuclei of somatic cells into the nucleus-free oocyte.

Another aspect provides a method of increasing the efficiency of embryo development or embryo formation, including culturing an oocyte in a medium containing melatonin. The specific content of the medium containing melatonin is as described above.

In one embodiment, the method may comprise contacting the oocyte with an agent that reduces H3K9me3 methylation. The details of the agent for reducing H3K9me3 methylation are as described above. In addition, the oocyte may be frozen and thawed. In addition, the oocyte may be a somatic cell nuclear transfer embryo.

The method may include incubating the oocyte for 1 to 10 days in a medium containing melatonin. Specifically, it may be cultured for 1 to 10 days, for 1 to 8 days, for 1 to 7 days, for 2 to 8 days, for 2 to 6 days, or for 3 to 6 days. If the incubation period is less than the above range, there is a problem that the embryo is not enough to develop into a blastocyst. If the incubation period is over the range, it is difficult to obtain an improved blastocyst because the oocyte is over maturated.

In addition, the method may further comprise the step of developing the embryo obtained in the culturing step as an individual. The subject may be a morula, a blastula and / or a gastrula.

Another aspect provides an embryo, blastocyst and / or embryonic stem cell produced by the method. Another aspect provides a graft composition comprising the embryo and / or the blastocyst produced by the above method as an active ingredient.

In another aspect, there is provided a method of preparing an oocyte, The present invention also provides a method for increasing the efficiency of somatic cell nuclear transfer (SCNT) comprising culturing the somatic cell nucleus and nucleus-free oocyte in a medium containing melatonin. The efficiency may be the success rate of the somatic cell nuclear transfer, or the embryo development rate of the cell generated by the somatic cell nuclear transfer. The embryo development rate may be the development into the blastocyst through the 2-cell, 4-cell and 8-cell groups of the embryo.

The present inventors confirmed that injection of KDM4A mRNA prevents the developmental arrest at the time of zygotic gene activation (ZGA) of the oocyte inducing KDM4A overexpression, that is, the 2-cell cycle, and significantly improves somatic cell nuclear transfer embryo development Respectively. Thus, the utility of oocyte donors for somatic cell nuclear transfer is expanded and the production of nuclear-grafted ESCs for both therapeutic replication and nuclear-grafted embryonic stem cell (NT-ESC), especially therapeutic applications and research and disease modeling May be used in a method of enhancing somatic cell nuclear transfer. The present invention is not intended for human reproductive cloning.

The method comprises the steps of removing the nuclei of oocytes, implanting the nuclei of one or more somatic cells (donor nuclei), activating the reconstructed nuclear transfer oocytes (embryo), and further culturing with a blastocyst As shown in FIG. Steps for such somatic cell nuclear transfer (SCNT) can be performed by a person skilled in the art according to a method disclosed in Nature 419, 583-587, 10 October 2002, etc., as appropriate. The nuclear transplanted oocyte (embryo) is generated from injecting donor somatic cell-derived nuclei (for example, nuclear genetic material) into the nucleus-deficient recipient oocyte to form nuclear-grafted oocyte, To form a fused, nuclear-engrafted embryo.

As used herein, the term "somatic cell" refers to a plant or animal cell that is not a germ cell or a germ cell precursor. The somatic cells are selected from the group consisting of cumulus cells, epithelial cells, fibroblasts, neurons, keratinocytes, hematopoietic cells, melanocytes, cartilage cells, erythrocytes, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, A cell, a fetal cell, and an adult cell.

As used herein, the term " somatic cell nuclear transfer "or" somatic cell nuclear transfer "refers to a technique of transplanting nuclei harvested from donor cells into donor cells that have been removed from the nucleus, .

The nucleus-free oocyte may be cumulus-oocyte complexes collected from the follicles of a subject, or may be obtained commercially. The subject may be a mammal, including a human. For example, the mammal may include rats, pigs, sheep, dogs, cows, horses, chlorine, and the like. In one embodiment, a somatic cell nuclear transfer embryo (SCNT-FOC) using oocyte cytoplasm (FOC) and a cytoplasm of freezing / thawed (low temperature preserved / thawed) SCNT-CROC), Kdm4a mRNA was injected, and the development rate of blastocyst was about 17% lower in SCNT-CROC (Fig. 2C). Melatonin enhanced the blastocyst development rate of SCNT-CROC separately from Kdm4a mRNA, and melatonin improved the blastocyst development rate only by injection of Kdm4a mRNA The developmental efficiency of the uninvolved blastocyst was improved by about 16%, confirming the development rate of blastocyst similar to SCNT-FOC (Table 2). Thus, the oocyte removed from the nucleus may be frozen and / or thawed or melted after cryopreservation. As the above-mentioned freezing method, slow-freezing or vitrification which is a quick freezing method can be used. Since melatonin-treated SCNT-CROC shows a blastocyst development rate similar to that of SCNT-FOC, it can be used for nuclear transplantation without any additional freezing and thawing of the donor oocyte during the somatic cell cloning process, which can shorten the process of somatic cell nuclear transfer And the replication efficiency can be improved.

The method according to one embodiment may include a step of culturing the somatic cell nucleus and the nucleus-free oocyte in vitro to form a blastocyst.

The method according to one embodiment may comprise contacting the agent with H3K9me3 methylation reducing agent. The method according to another embodiment may include injecting a formulation that reduces H3K9me3 methylation. The agent that reduces H3K9me3 methylation may be one that increases the expression of members of the KDM4 family of histone demethylating enzymes. The agent may be one that increases the expression or activity of, for example, KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD2D) or a combination thereof. The contacting or implanting step may be performed on the nuclear transfer embryo after the nucleus of the somatic cell and the oocyte removed from the nucleus are fused. Specifically, the embryo may be an embryo before activation of a somatic cell nuclear transfer oocyte gene. For example, the embryo may be incubated for 5 hours post activation (5 hpa) or between 10 and 12 hpa (i. E., At the 1-cell stage), or at about 20 hpa Or between 20 and 28 hpa (i. E., 2-celled) with one or more histone demethylating enzyme KDM4 family.

In addition, the step of contacting or injecting may include contacting or donating one or more histone demethylase KDM4 families to a donor cell, e. G., A terminally differentiated somatic cell nucleus or cytoplasm, prior to injecting the donor cell nucleus into the nucleated oocyte. It can be injected. The contact or infusion may be contact with the donor somatic cell for at least 1 hour, or more than 2 hours, and the contact may be from 1 day (24 hours) to 2 days prior to removal of the nucleus from the donor somatic cell Day, more than three days, or more than three days.

Therefore, by contacting the somatic cell nucleus and nucleus-removed oocyte with an agent for reducing H3K9me3 methylation, the activity of the histone methylase which inhibited replication in the somatic cell nuclear transfer embryo is reduced, and the expression of the embryo-related gene is promoted Bar, and blastocyst development efficiency can be improved. For example, the developmental efficiency may be an increase in the percentage of development of a somatic cell nuclear transfer embryo developing into a 2-cell, 4-cell and 8-cell or blastocyst stage and may be an 8-cell or blastocyst stage of somatic cell nuclear transfer embryo Can increase the pre-implant development efficiency. That is, the agent for reducing the H3K9me3 methylation can overcome the 2-cell stopping phenomenon in the somatic cell nuclear transfer embryo and can maintain the continuous embryonic development.

The method of one embodiment may include incubating the nucleus of the somatic cell and the oocyte removed from the medium for 1 to 10 days in the medium containing the melatonin. Specifically, it may be cultured for 1 to 10 days, for 1 to 8 days, for 1 to 7 days, for 2 to 8 days, for 2 to 6 days, or for 3 to 6 days. If the incubation period is less than the above range, there is a problem that somatic cell nuclear transfer embryos are not enough to develop into blastocysts. If the incubation period is over the range, somatic cell nuclear transfer embryos are over-matured and qualitatively improved blastocysts There is a problem that it is difficult.

In one embodiment, the method further comprises administering to the subject an effective amount of at least about 5%, at least about 10%, at least about 13%, at least about 15%, at least about 15% , Greater than about 30%, greater than about 50%, greater than 50 to 80%, or greater than 80%. That is, it is possible to increase the efficiency of the pre-implantation development of a somatic cell nuclear transfer embryo, or to increase the development of the embryo to the blastocyst stage or to increase the development of the embryo to the expanded blastocyst stage by about 5%, about 7% %, About 12 or more, or more than 12%. In another embodiment, the method is characterized in that the successful development into the blastocyst stage is more than 3-fold, 4-fold, 5-fold, 6-fold, or more than the somatic cell nuclear transfer performed in the absence of the agent that reduces H3K9me3 methylation. 7-fold, 8-fold, or 8-fold increase. An increase in the somatic cell nuclear transfer efficiency means an increase or an increase in the production of blastocysts. The increase in the production or the yield of the blastocyst is greater than or equal to about 110%, greater than about 120%, greater than about 130%, less than about 140%, or less than about 140%, compared to somatic cell nuclear transfer performed in the absence of agents that reduce H3K9me3 methylation. , More than about 150%, or more than about 150%.

As described above, the method according to one aspect of the present invention can prevent cell damage by reducing the active oxygen of somatic cell nuclear transfer cells by culturing somatic cell nuclear transfer cells in a medium containing melatonin, Can be improved. In addition, the cell death of somatic cell nuclear transfer embryos frozen and melted by melatonin is reduced, which can improve blastocyst formation and production efficiency. In addition, as the implantation rate of the somatic cell nuclear transfer embryo is improved, it is possible to produce an endangered animal through in vitro fertilization. As the induction of embryonic stem cells is promoted, the embryonic stem cell line can be efficiently produced.

Another aspect provides a somatic cell nuclear transfer embryo made according to the method. Another aspect provides a somatic cell nuclear transfer blastocyst prepared according to the method. The embryo is genetically modified, and can be modified, for example, prior to somatic cell nuclear transfer (i. E., Prior to donor nuclei collection and fusion with the cytoplasm of the recipient oocyte) Lt; / RTI > In one embodiment, the embryo may comprise nuclear DNA derived from donor somatic cells, cytoplasm derived from recipient oocytes, and mitochondrial DNA derived from a third donor individual. The embryo or blastocyst is characterized in that the expression of a gene associated with cell survival, tissue regeneration, antioxidant function, inflammation or apoptosis is upregulated or downregulated compared to a somatic cell nuclear transfer embryo or blastocyst performed in the absence of an agent that reduces H3K9me3 methylation Lt; / RTI >

In one embodiment, the embryo may be one in which expression of a gene involved in cell survival and tissue regeneration is upregulated. Specifically, the gene may be Amd1 , Fam46C , Oxt, and / or Ppt2 . In addition, the expression of genes involved in antioxidant function, inflammation or apoptosis may be upregulated. Specifically, the gene may be Ctss , Dffa , Eif3f , Hacl1 , Hspbp1 , Mob2 , Mrpl23 , Mrpl33 , Pmvk , Slc4a11 And / or Wbscr22 . ≪ / RTI > Also, cell death or degeneration related genes such as Atp6v0c, Cd52 , Dapk2 , Ddit4l , Duoxa2 , Nfkbia , Ptgds , Rdh12 And / or the expression of Rnd3 is down-regulated.

In other embodiments, the blastocyst may be up-regulated in expression of genes associated with cell survival or tissue regeneration, such as Amd1 , Fam46C , Oxt , and / or Ppt2 . In particular, they are genetics related to antioxidant function, inflammation or apoptosis, for example, Gulo And / or the expression of Txnip is upregulated. On the other hand, genes related to oxidative stress, such as Adh1 , Car2 , Gsta3 , Gstm2 , Mb , Phlda2 , and / or S100a1 ; The expression of apoptosis-related genes such as Akr1c13 , Amd2 , Clu , Hist1h3a , Hspb1 , Il1rn, and / or Nat8 may be down-regulated. Also, genes associated with cell and tumor proliferation, such as Efempl , Gliprl , Lgals4 , Plac1 , Snora15 , Snora21, Snora34 , and / or Anxa1 ; The expression of genes related to the immune response, such as Clec2f , Hrsp12, and / or Plat may be downregulated.

Another aspect provides somatic cell nuclear transfer embryonic stem cells prepared according to the above method. The embryonic stem cells are obtained by separating the cells from the inner cell mass in a blastocyst prepared according to the method; And culturing the undifferentiated inner cell mass-derived cells. In addition, the embryonic stem cells may be pluripotent stem cells or pluripotent stem cells.

In another aspect, there is provided a method of preparing an oocyte, Culturing the somatic cell nucleus and the oocyte from which the nucleus has been removed together with the candidate substance; And evaluating the H3K9me3 methylation activity of the somatic cell nuclear transferring cells generated after the culturing, the method comprising screening the agent for increasing somatic cell replication efficiency.

Cells derived from somatic cell nuclear transfer embryos or blastocysts, such as embryonic stem cells, may be used in a test to determine whether the agent 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 agent and can be used for screening to select agents that affect cells derived from somatic cell nuclear transfer embryos or blastocysts . The test compound may be any compound of interest, including chemical compounds, small molecules, polypeptides or other biological agents (e. G., Antibodies or cytokines, etc.).

In the method according to one embodiment, the step of evaluating the H3K9me3 methylation activity is to determine the degree of expression of a gene or protein that reduces H3K9me3 methylation, using techniques well known in the art such as RT-PCR or immunostaining . In addition, the step of evaluating the H3K9me3 methylation activity may be performed together with the step of evaluating the somatic cell replication efficiency. If the number of somatic cell nuclear transfer oocyte, 2-cell stage cell, 4-cell stage cell or blastocyst stage cell number in the culture after addition is changed as compared with before the addition of the candidate substance, the candidate substance may be the H3K9me3 methylation It is possible to determine that it is a substance which decreases activity and further increases the efficiency of somatic cell replication by somatic cell nuclear transfer.

In another aspect, there is provided a method of preparing an oocyte, Obtaining somatic-cell nuclear transfer (SCNT) cells by culturing oocytes in which the nuclei and nuclei of the somatic cells are removed in a medium containing melatonin; And a step of in vitro fertilization of the somatic cell nuclear transfer cells and the liquid semen of the somatic cell transplantation embryo. The details of the method of culturing the oocyte in which the nucleus and nucleus of the somatic cell are removed in a medium containing melatonin are as described above. The somatic cell nuclear transfer cell may be a somatic cell nuclear transfer embryo, specifically, a cell line of a 2-cell line, a 4-cell line and an 8-cell line, and may be a cell that has developed into the blastocyst stage and has reached the blastocyst stage.

The method according to one embodiment comprises in vitro fertilization of the somatic cell nuclear transfer cell and the liquid semen. The semen may be a semen taken from the body of the subject. The subject may be a mammal, including a human. For example, the mammal may include rats, pigs, sheep, dogs, cows, horses, chlorine, and the like. The somatic cell nuclear transfer cell and the liquid semen may be in vitro fertilized in the in vitro fertilization medium composition for 1 to 7 days. At this time, when the IVF period is less than the above range, there is a problem that it can not be corrected, and when it exceeds the above range, the embryo is degraded. In addition, the step may further comprise contacting or injecting with an agent that reduces H3K9me3 methylation. Specific details of the agent for reducing the methylation are as described above. The methylation-decreasing agent may be added to somatic cell nuclear transfer embryos after the injection of semen, before the formation of nucleus (18 hours after semen injection), specifically injecting semen, It can be injected. Therefore, the somatic embryo-transferred embryo is successfully developed into a blastocyst through the 2-, 4-, and 8-cell stage without progression of the somatic cell nuclear transfer embryo (embryo), development defect, or loss of viability, Can be increased.

The present invention relates to a composition for embryo development comprising melatonin and a method for improving the embryo development rate using the same, wherein the cell is efficiently developed into a blastocyst without cell damage and / or developmental arrest, Can be used to produce somatic cell cloned embryos.

1A is a photograph showing the results of observing demethylation of H3K9me3 by injecting Kdm4a mRNA into SCNT-FOC and SCNT-CROC groups.

FIG. 1B is a graph showing the results of observing demethylation of H3K9me3 by injecting Kdm4a mRNA into SCNT-FOC and SCNT-CROC groups.

FIG. 2A is a photograph showing changes in the number of cells and whole cells of SCNT-FOC and SCNT-CROC groups according to whether Kdm4a mRNA was injected.

FIG. 2B is a photograph showing the embryo development level of SCNT-FOC and SCNT-CROC groups according to whether Kdm4a mRNA was injected.

FIG. 2C is a graph showing the blastocyst quality ratio of SCNT-FOC and SCNT-CROC groups according to whether Kdm4a mRNA is injected.

FIG. 3A shows genes having different expression patterns in SCNT-FOC and SCNT-CROC group 2-cell embryos according to whether Kdm4a mRNA was injected.

FIG. 3B shows genes regulated (up / down) in SCNT-FOC and SCNT-CROC group 2-cell embryos according to the injection of Kdm4a mRNA.

FIG. 4A is a photograph showing the DNA fragmentation level in blastocysts of SCNT-CROC and SCNT-CROC + K groups with and without melatonin treatment by TUNEL staining.

FIG. 4B is a graph showing the number of TUNEL-positive cells in the blastocysts of SCNT-CROC and SCNT-CROC + K groups with and without melatonin treatment.

FIG. 5A is a photograph showing fluorescence staining (cell death, green, nucleus, blue staining) of a site where apoptosis has occurred in the SCNT-CROC + K group with or without melatonin treatment.

FIG. 5B is a graph showing the levels of reactive oxygen species in the supernatant of SCNT-CROC + K group with and without melatonin treatment.

6A is a photograph visualizing the implantation of the SCNT-CROC + K group in the mouse uterus according to the presence or absence of the melatonin treatment.

FIG. 6B is a photograph showing a result of repeatedly experimenting whether or not the SCNT-CROC + K group is implanted in the mouse uterus according to the presence or absence of the melatonin treatment.

6C is a graph showing the embryo transfer rate of the SCNT-CROC + K group according to the presence or absence of the melatonin treatment.

FIG. 7A is a result of microscopic examination of the mESC induction rate of SCNT-CROC + K group depending on whether or not the melatonin treatment is carried out.

FIG. 7B is a graph showing the mESC induction rate of the SCNT-CROC + K group according to the presence or absence of the melatonin treatment.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Preparation example]

Female B6D2F1 mice (Orient-bio, Gyeonggi-do, Korea) were used at 8-10 weeks of age to collect recipient oocytes and SCNT donor oocytes. 8- to 10-week-old female ICR mice were used as surrogate mothers of embryo transfer. To induce pseudopragnancy, the mice were crossed with male mice of the same strain. The protocol for animal use in this study was approved by the Committee on Animal Experimentation and Use (IACUC) (project number IACUC-170119) of the University of Science in Caracas, and all experiments were performed according to an approved protocol.

[Example]

Example 1. Collection of oocytes

5 IU of pregnant mare serum gonadotropin (PMSG; Sigma-Aldrich, St. Louis, Mo.) was injected into the mice of the preparation example and after 48 hours, 5 IU of human chorionic gonadotropin gonadotropin, hCG; Sigma-Aldrich) to induce superovulation. Oocytes were collected in M2 (Sigma-Aldrich) medium after 14 hours of hCG injection in mice and the cumulus cells were removed with a medium containing 0.1% hyaluronidase (Sigma-Aldrich) (Denuded). Cumulus-free oocytes were then cultured in potassium simplex optimized medium (KSOM, Millipore, Darmstadt, Germany) for the experiment. The dispersed cumulus cells were treated with hyaluronidase The pellet was resuspended in M2 in a small volume of polyvinylpyrrolidone (PVP) 3% (v / v) and stored at 4 ° C until use.

Example 2. Vitrification of oocytes Vitrification and warming

Quinn 'with 20% (v / v) HEPES (Sage, Malov, Denmark) as knockout serum replacement (KSR, Gibco, Grand Island, NY) as the primary medium for vitrification and preparation of warming solution. Were used. Vitrified with two cryoprotectant agents, a combination of ethylene glycol (EG, Sigma-Aldrich) and dimethylsulfoxide (DMSO, Sigma-Aldrich). MII oocytes were pre-equilibrated for 2 minutes 30 seconds with HEPES medium containing 7.5% ethylene glycol and 7.5% DMSO. Pre-equilibrated oocytes were equilibrated for 20 seconds in the same volume of HEPES medium supplemented with 15% ethylene glycol, 15% DMSO, and 0.5 M sucrose (Sigma-Aldrich). Equilibrated oocytes were loaded into electron microscopic copper grids (EM Grid, PELCO, Redding, Calif.) And slush nitrogen (SN2) was eluted using Vit-master (IMT, Ness Ziona, Israel) Lt; / RTI > The vitrified frozen oocytes were stored in the LN2 tank. For warming, the vitrified frozen oocytes were warmed in four steps. The EM grid was sequentially transferred to 0.5, 0.25, 0.125, and 0 M sucrose at 37 < 0 > C at 2 min 30 sec intervals. The oocytes were then washed three times with modified HTF (Millipore) medium and cultured in HTF medium until the start of the experiment.

Example 3: Somatic cell nuclear transfer (SCNT)

Fresh or vitrified vitrification / warmed oocytes prepared in Example 2 were incubated at 37 ° C, and the oocytes were removed from the oocytes. Fresh cumulus cells were used as nuclear donor oocytes. The nuclei of fresh, vitrified, frozen / thawed cumulus cells were removed in M2 medium containing 5 μg / ml of cytochalasin B. For nuclear transfer, cumulus cells were injected into enucleated oocytes using a piezo-driven micromanipulator (Primetech LTD, Tsuchiura-shi, Japan) in M2 medium. Subsequently, embryos regenerated with 10 mM SrCl ₂ , 2 mM EGTA, and 5 μg / ml cytochalasin _B in M16 (Millipore) medium were activated for 6 hours and then incubated at 37 ° C in a humidified atmosphere of 5% CO ₂ Of KSOM. The somatic cell oocyte group using fresh oocyte cytoplasm (FOC) was named as SCNT-FOC and the group using cytoplasm of vitrified / frozen (low temperature preserved / thawed) oocyte was named SCNT-CROC Respectively.

Example  4. Kdm4a mRNA  Embryo development and transcriptional differences of somatic cell nuclear transfer

4-1. Kdm4a  Preparation of mRNA

Full-length mouse Kdm4a / Jhdm3a cDNA was cloned into a pcDNA3.1 plasmid containing poly (A) 83 at the 3 'end of the cloning site using an In-Fusion Kit (Clonetech # 638909). MRNA was synthesized from a template plasmid linearized by in vitro transcription 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); Aliquots of mRNA were stored at -80 ° C until use.

4-2. Determination of somatic cell nuclear transfer embryo development

The injection of Kdm4a mRNA encoding the H3K9me3 demethylase is known to improve oocyte development in somatic cell nuclear transfer mammals. First, to evaluate the effect of Kdm4a mRNA on the development of cloned oocytes (SCNT-CROC) using freezing / thawing cytoplasm of freezing / thawing oocytes, embryonic development was evaluated regardless of whether Kdm4a mRNA was injected or not. Next, ~10 pl of water (control) or 2 / / ㎕ Kdm4a mRNA was injected into the two groups of oocytes (SCNT-FOC group and SCNT-CROC group) prepared in Example 3 using a piezo-driven micromanipulator Respectively. In addition, embryo development was confirmed by immunofluorescence. Specifically, the embryos cloned with phosphate-buffered saline (PBS) containing 0.1% polyvinyl alcohol (PVA) were washed and incubated with 4% w / v paraformaldehyde at room temperature. For 30 minutes. The oocytes were washed with PBS / PVA and incubated overnight at 4 ° C in PBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100. The oocytes were then washed three times in PBS-0.1% BSA and incubated for 2 hours with 1: 200 dilution of H3K9me3 antibody and purified mouse anti-OCT-3/4 (BD) at 1 and 2 cell steps. Cloned embryos were washed three times with PBS-0.1% BSA and incubated with 1: 200 dilution of goat anti-mouse antibody for 1 hour at room temperature. Thereafter, the cells were further washed three times with PBS-0.1% BSA. DNA was visualized by staining oocytes with 4 ', 6-diamidino-2-phenylindole (DAPI). The embryos were placed on glass slides with fluorescent mounting media and observed using a fluorescence microscope (Zeiss LSM 880, Zeiss, Jena, Germany).

group	mRNA (μg / μl)	Nuclear transfer oocyte number	Number of embryos in 2-cell stage (%) ¶	Number of 4-cell embryos (%) #	Number of 2-cell block embryos (% ± SEM) #	Number of blastocysts (% ± SEM) #
SCNT-FOC	-	112	105 (94)	70 (67)	35 (33 ± 2.9) a	27 (26 +/- 3.4) a
SCNT-FOC + K	2	109	103 (95)	102 (99)	1 (1 + 1) b	85 (83 ± 3.5) b
SCNT-CROC	-	139	132 (95)	93 (70)	39 (30 1.8) a	30 (23 ± 3.1) a
SCNT-CROC + K	2	130	125 (96)	123 (98)	2 (2 + - 1.2) b	82 (66 +/- 2.4) c

a, b, c: Significant difference in value (P <0.05; n = 5) as another superset within the same column

SCNT-FOC: Somatic cell nuclear transfer cloned oocyte using cytoplasm of fresh oocyte

SCNT-CROC: Somatic cell nuclear transfer cloned oocyte using cytoplasm of freezing / thawed oocyte

K: injection of lysine (K) -specific demethylating enzyme 4A (Kdm4a) mRNA

¶: Based on the number of SCNT oocytes

#: Based on the number of 2-cell embryos

Figure 1 shows the results of demethylation of H3K9me3 by injecting Kdm4a mRNA into SCNT-FOC and SCNT-CROC groups. Table 1 shows the effect of Kdm4a mRNA injection on the development of SCNT-FOC and SCNT-CROC groups to be. It was confirmed that demethylation of H3K9me3 was normally performed in the SCNT-FOC group and the SCNT-CROC group 1 cell group and the nucleus of the second cell group (Fig. 1A). As a result of quantifying the methylation level, it was confirmed that the level of H3K9me3 was significantly decreased when Kdm4a mRNA was injected into SCNT-FOC group and SCNT-CROC (FIG. 1B). In addition, when Kdm4a mRNA was injected into the SCNT-FOC group and the SCNT-CROC group, the blastocyst efficiency of both groups was significantly increased. In other words, it was confirmed that down-regulation of H3K9me3 activity improves both blastocyst formation rates of the embryos of SCNT-FOC group and SCNT-CROC group. Thus, the injection of Kdm4a mRNA inhibits H3K9me3 activity and overcomes the 2-cell block in the oocyte (Table 1).

FIG. 2 shows changes in the number of cells and whole cells of SCNT-FOC and SCNT-CROC groups according to whether Kdm4a mRNA was injected. At this time, the inner cell group means the number of cells capable of developing into embryonic stem cells in the future, and Type 1 indicates less than 10 intracellular cells and Type 2 indicates 10 or more intracellular cells.

As shown in FIGS. 2A and 2B, embryo development defects in the SCNT-FOC group and the SCNT-CROC group were substantially overcome after the injection of the mRNA, but the embryo development and embryo development degree in the SCNT-CROC group were significantly Of the embryo development and development. In particular, as shown in FIG. 2C, the number of cloned embryos having a high ratio of ICM number (more than 10 ICMs per blastocyst) in the SCNT-CROC + K group was significantly smaller than that in the SCNT-FOC + K group (p <0.05) . In other words, the injection of Kdm4a mRNA significantly improves the quality of blastocysts by significantly increasing the number of cell population.

4-3. Identification of transcription of 2-cell embryos in somatic cell nuclear transfer cells

To determine the transcriptional differences of the earliest embryo between SCNT-FOC and SCNT-CROC groups, two groups of pooled 2-cell embryos (100 embryos per sample, 24 h after embryo activation and Kdm4a mRNA injection, 3 &lt; / RTI > cycles). Gene expression was assessed in a 2-cell embryo between SCNT-FOC + K and SCNT-CROC + K (100 embryos per sample, 3 replicates). In the second round, gene expression was evaluated in blastocysts between SCNT-CROC + K and SCNT-CROC + K + M (50 blastocysts per sample, 2 replicates). The complementary DNA (cDNA) was amplified using the SMARTer ultra low input RNA cDNA preparation kit (Takara, 634890) according to the manufacturer's instructions. CDNA was fragmented into approximately 200 bp fragments using an M220 sonicator (Covaris). The fragmented cDNA was end-repaired and adapter-ligated. The sequencing library was prepared using the ScriptSeq v2 kit (Illumina) according to the manufacturer's instructions. Single-end sequencing was performed on HiSeq2500 (Illumina) and mapped to the mm9 mouse genome using STAR (v2.5.2b, https://github.com/alexdobin/STAR). We then calculated FPKM (fragments per kilobase per million read) with Cufflinks (v2.2.1) using the default option. The genes were considered to be differentially expressed in fold change> 3 and FPKM> 5. Statistical analysis and scatter plot generation were performed using the R (v3.3.2) package. Expression of most genes was similar in both groups and only a few genes (159) showed different expression patterns (Fig. 3a). In GO terms and KEGG analysis, several pathways including cell death and p53 signaling pathway were found to be significantly activated. When DEG was analyzed in the GO terms-database, only 16 genes (0.92%) among 1,730 apoptosis-related genes showed more than 3-fold difference between SCNT-FOC group and SCNT-CROC group. In the SCNT-CROC group, eight pro-apoptotic genes (Cyt, Dapk2, Dffb, Gadd45g, Hint2, Mien1, P2rx7 and Pmaip) and three anti-apoptotic genes (Adrb2, Scin, The expression was up-regulated compared to the SCNT-FOC group. In addition, it was confirmed that only two apoptosis-related genes (Ifng and Siah1a) (18.2%) and three anti-apoptotic genes (Syce3, Tsc22d3 and Vegfa) (27.2%) were down-regulated.

In SCNT-CROC group, only 6 genes (0.41%) among 1,449 cell cycle-related genes showed different expression levels and one cell arrest related gene (Gadd45g) was upregulated (FIG. 3B). On the other hand, in SCNT-FOC group and SCNT-CROC group, 30 gene expression related genes (0.62%) among 4,841 genes and 61 genes (0.60%) among 10,234 metabolic pathway related genes were different from each other there was. However, most of the genes do not function negatively in gene expression or metabolism (FIG. 3B).

Example 5 Embryonic development of somatic cell cloned oocytes with and without melatonin

5-1. Embryonic development of somatic cell nuclear transfer cells with or without melatonin

Kdm4a (SCNT-CROC + K) was first injected into the SCNT-CROC group oocytes and then cultured in KSOM medium containing 10 μM of melatonin (Sigma) and KSOM medium not containing KMOM. Embryonic development of cloned oocytes was assessed for 5 days (120 hours) after activation. The melatonin concentration was selected through preliminary experiments using conventional fertilized mouse embryos at concentrations showing the highest quality and high blastocyst formation rates. The results for each group are shown in Table 2 below.

group	Melatonin (M)	Nuclear transfer oocyte number	Number of embryos in 2-cell stage (%) ¶	Number of 4-cell embryos (%) #	Number of 2-cell block embryos (% ± SEM) #	Number of blastocysts (% ± SEM) #
SCNT-CROC	-	125	112 (90)	66 (59)	46 (42 ± 3.7) a	23 (20 ± 1.5) a
SCNT-CROC + M	10	125	114 (91)	81 (71)	33 (31 ± 3.7) a	44 (39 ± 1.2) b
SCNT-CROC + K	-	138	128 (98)	124 (94)	4 (3 + 1.5) a	76 (59 +/- 3.3) a
SCNT-CROC + K + M	10	138	123 (96)	119 (96)	4 (4 + 1.8) a	91 (75 ± 3.3) b

a, b, c: Significant difference in value (P <0.05; n = 5) as another superset within the same column

SCNT-FOC: Somatic cell nuclear transfer cloned oocyte using cytoplasm of fresh oocyte

SCNT-CROC: Somatic cell nuclear transfer cloned oocyte using cytoplasm of freezing / thawed oocyte

M: Melatonin treatment

K: injection of lysine (K) -specific demethylating enzyme 4A (Kdm4a) mRNA

¶: Based on the number of SCNT oocytes

#: Based on the number of 2-cell embryos

As a result, melatonin played a positive role in the embryonic development of the SCNT-CROC group regardless of the injection of Kdm4a mRNA (Table 2). That is, treatment of melatonin improves the development rate of blastocysts of SCNT-CROC group separately from Kdm4a mRNA injection. In addition, it was confirmed that the development rate of blastocyst development similar to that of SCNT-FOC group was confirmed by increasing the efficiency of blastocyst development, which was not overcome by injection of Kdm4a mRNA into SCNT-CROC group.

5-2. Confirmation of DNA Fragmentation by Melatonin Treatment

The DNA fragmentation level in the blastocysts according to whether melatonin treatment was performed in the group of Example 5-1 was detected using TUNEL staining method ( in situ Cell Death Detection Kit, Roche, Indianapolis, Ind.). SCNT-derived blastocysts were washed three times in DPBS 0.1% PVA. The embryos were incubated in TUNEL reaction medium at 37 &lt; 0 &gt; C, dark for 1 hour. DNA stained with 1 [mu] g / ml Hoechst 33342 (Bis-benzimide, Sigma) was used for nuclear counter-staining. The embryonic signal was observed with a confocal microscope (Zeiss, LSM880).

As a result, it was confirmed that the treatment of melatonin reduced the number of TUNEL-positive cells in the blastocyst of the SCNT-CROC group (Fig. 4A). The overall percentage of TUNEL-positive embryos was significantly reduced in the melatonin treated group (SCNT-CROC + K + M) (7.8% vs. 54.3% and 24.1%) compared to the melatonin untreated group (SCNT-CROC and SCNT-CROC + K) %) (p <0.05; Fig. 4b).

5-3. Effects of melatonin treatment on the production of free radicals in embryos

The effect of the melatonin treatment on the active oxygen of the blastocysts in the group of Example 5-1 was confirmed. Specifically, in the SCNT-CROC + K group, embryos at the blastocyst stage or blastocyst stage were treated with melatonin or without melatonin and cultured for 30 minutes in a culture medium containing 5 μM CellROX oxidative stress reagent (Molecular Probes, Eugene, OR) , And then treated with 0.1% polyvinyl alcohol (PVA) -D-PBS. Embryos were observed with a fluorescence confocal microscope (Zeiss, LSM880). The recorded fluorescence intensity was analyzed using ImageJ software. Fluorescent pixel values of the embryos were measured within a certain region from the cytoplasmic region of another embryo. Before analysis of statistically significant differences between groups, background fluorescence values were excluded from the final values. Five to ten embryos were replicated three times in each replicate.

As a result, it was confirmed that the treatment with melatonin significantly reduced the level of free radicals in the blastocyst. In addition, the effect of treatment with melatonin on the quality of blastocysts through fluorescence staining (cell death, green, nuclear, and blue dyeing) of apoptotic parts was significantly reduced And the cell death rate was also significantly decreased (FIG. 5A). In addition, it was confirmed that the level of active oxygen in the supernatant was significantly lower in the SCNT-CROC group than in the SCNT-CROC + K group (Fig. 5B). That is, the treatment of melatonin improves the apoptosis pathway such as apoptosis, peroxisome and oxidative phosphorylation, so that a qualitatively improved blastocyst can be obtained.

5-4. Melatonin treatment Blastocyst  Identify the impact on the warrior

To confirm which genes were regulated by melatonin, transcripts of 2-cell embryos and blastocysts (50 embryos per embryo or blastocyst, twice) in both the melatonin treated and untreated groups of Example 5-1 And the results are shown in Table 3 below.

Related features	Gene symbol	Gene ID	Gene name	control
Cell survival and tissue regeneration	Deaf1	NM_001282072.1	DEAF1, transcription factor	Standing
	Fxn	NM_008044.2	frataxin	lift
	Ppan	NM_145610.2	peter pan homolog	lift
	Rab10os	NR_015551.1	RAB10, member RAS oncogene family, opposite strand	lift
	Sprr2d	NM_011470.2	small proline-rich protein 2D	lift
	Stag3	NM_016964.2	stromal antigen 3	lift
	Tsen15	NM_025677.3	tRNA splicing endonuclease subunit 15	lift
	Zfp335	NM_199027.2	zinc finger protein 335	lift
	Adm	NM_009627.2	adrenomedullin	Downward
	Zfp54	NM_011760.2	zinc finger protein 54	Downward
	Zscan4f	NM_001110316.2	zinc finger and SCAN domain containing 4F	Downward
Antioxidant, inflammation and cell death	Ctss	NM_001267695.2	cathepsin S	lift
	Dffa	NM_001025296.2	DNA fragmentation factor, alpha subunit	lift
Oxidative stress	Eif3f	NM_025344.2	eukaryotic translation initiation factor 3, subunit F	lift
	Hacl1	NM_019975.3	2-hydroxyacyl-CoA lyase 1	lift
	Hspbp1	NM_001360629.1	HSPA (heat shock 70kDa) binding protein, cytoplasmic cochaperone 1	lift
	Mob2	NM_001347560.1	MOB kinase activator 2	lift
	Mrpl23	NM_011288.1	mitochondrial ribosomal protein L23	lift
	Mrpl33	NM_025796.3	mitochondrial ribosomal protein L33	lift
	Pmvk	NM_001310640.1	phosphomevalonate kinase	lift
	Slc4a11	NM_001081162.1	solute carrier family 4, sodium bicarbonate transporter-like, member 11	lift
	Wbscr22	NM_001202560.2	BUD23, rRNA methyltransferase and ribosome maturation factor	lift
Cell death / regeneration	Ctsd	NM_009983.3	cathepsin D	lift
	Ifi27	NM_001364173.1	interferon, alpha-inducible protein 27	lift
	Mettl13	NM_144877.1	methyltransferase like 13	lift
	Nck1	NM_001324530.1	non-catalytic region of tyrosine kinase adapter protein 1	lift
	Nmi	NM_001141948.1	N-myc (and STAT) interactor	lift
	Atp6v0c	NM_001361531.1	ATPase, H + transporting, lysosomal V0 subunit C	Downward
	Cd52	NM_013706.2	CD52 antigen	Downward
	Dapk2	NM_010019.3	death-associated protein kinase 2	Downward
	Ddit4l	NM_030143.4	DNA-damage-inducible transcript 4-like	Downward
	Duoxa2	NM_025777.2	dual oxidase maturation factor 2	Downward
	Nfkbia	NM_010907.2	nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha	Downward
	Ptgds	NM_008963.2	prostaglandin D2 synthase (brain)	Downward
	Rdh12	NM_001313971.1	retinol dehydrogenase 12	Downward
	Rnd3	NM_028810.2	Rho family GTPase 3	Downward
Cell and tumor proliferation	AY074887	NM_145229.2	cDNA sequence AY074887	lift
	Cdca2	NM_001110162.1	cell division cycle associated 2	lift
	Dnph1	NM_207161.3	2'-deoxynucleoside 5'-phosphate N-hydrolase 1	lift
	Eif3d	NM_018749.2	eukaryotic translation initiation factor 3, subunit D	lift
	Eif3e	NM_008388.2	eukaryotic translation initiation factor 3, subunit E	lift
	Gtf2h2	NM_001360706.1	general transcription factor II H, polypeptide 2	lift
	Nefl	NM_010910.2	neurofilament, light polypeptide	lift
	Recql	NM_001204906.1	RecQ protein-like	lift
	Snhg7	NR_024068.2	small nucleolar RNA host gene 7	lift
	Ccne2	NM_001037134.2	cyclin E2	Downward
	Ly6a	NM_001271416.1	lymphocyte antigen 6 complex, locus A	Downward
Immune response	Ccdc86	NM_023731.3	coiled-coil domain containing 86	lift
	Commd4	NM_025417.2	COMM domain containing 4	lift
	Il17ra	NM_008359.2	interleukin 17 receptor A	lift
	Lat2	NM_020044.3	linker for activation of T cells family, member 2	lift
	Cstb	NM_007793.3	cystatin B	Downward
	Qpct	NM_001316729.1	glutaminyl-peptide cyclotransferase (glutaminyl cyclase)	Downward
Etc	Arfgap2	NM_001166024.1	ADP-ribosylation factor GTPase activating protein 2	lift
	Bcdin3d	NM_029236.2	BCDIN3 domain	lift
	Cenpl	NM_001127181.2	centromere protein L	lift
	Cep63	NM_001081122.1	centrosomal protein 63	lift
	Clpp	NM_017393.2	caseinolytic mitochondrial matrix peptidase proteolytic subunit	lift
	Cmtm7	NM_001252479.1	CKLF-like MARVEL transmembrane domain containing 7	lift
	Cox17	NM_001017429.2	cytochrome c oxidase assembly protein 17, copper chaperone	lift
	Dbnl	NM_001146308.1	drebrin-like	lift
	Defb22	NM_001002791.2	defensin beta 22	lift
	Dnaaf1	NM_026648.4	dynein, axonemal assembly factor 1	lift
	Dph2	NM_026344.3	DPH2 homolog	lift
	Elmod3	NM_001253692.1	ELMO / CED-12 domain containing 3	lift
	Gtsf1l	NM_026630.2	gametocyte specific factor 1-like	lift
	Hint3	NM_025798.3	histidine triad nucleotide binding protein 3	lift
	Ltc4s	NM_001313968.1	leukotriene C4 synthase	lift
	Ltn1	NM_001081068.1	listerine E3 ubiquitin protein ligase 1	lift
	Mars2	NM_175439.3	methionine-tRNA synthetase 2 (mitochondrial)	lift
	Meig1	NM_001355205.1	meiosis expressed gene 1	lift
	Mppe1	NM_001357518.1	metallophosphoesterase 1	lift
	Mbb12a	NM_028617.2	multivesicular body subunit 12A	lift
	Mvd	NM_138656.2	mevalonate (diphospho) decarboxylase	lift
	Ndufs7	NM_001364694.1	NADH: ubiquinone oxidoreductase core subunit S7	lift
	Nt5c	NM_015807.1	5 ', 3'-nucleotidase, cytosolic	lift
	Oxa1l	NM_026936.3	oxidase assembly 1-like	lift
	Pet100	NM_001195244.1	PET100 homolog	lift
	Psmd13	NM_011875.4	proteasome (prosome, macropain) 26S subunit, non-ATPase, 13	lift
	Psmg3	NM_001356963.1	proteasome (prosome, macropain) assembly chaperone 3	lift
	Ptpn18	NM_011206.2	protein tyrosine phosphatase, non-receptor type 18	lift
	Pxmp4	NM_021534.3	peroxisomal membrane protein 4	lift
	Slc35f5	NM_001356294.1	solute carrier family 35, member F5	lift
	Sln	NM_025540.2	sarcolipin	lift
	Srp68	NM_146032.3	signal recognition particle 68	lift
	Surf2	NM_013678.2	surfeit gene 2	lift
	Tagap1	NM_147155.2	T cell activation GTPase activating protein 1	lift
	Tbccd1	NM_001081368.2	TBCC domain containing 1	lift
	Tead4	NM_001080979.1	TEA domain family member 4	lift
	Tmem126b	NM_026734.1	transmembrane protein 126B	lift
	Tmem205	NM_001253867.1	transmembrane protein 205	lift
	Tor1b	NM_133673.3	torsin family 1, member B	lift
	Trmt61a	NM_001099792.1	tRNA methyltransferase 61A	lift
	Wrb	NM_207301.3	tryptophan rich basic protein	lift
	Ythdf1	NM_173761.3	YTH domain family 1	lift
	Zfp42	NM_009556.3	zinc finger protein 42	lift
	Lhfpl1	NM_178358.3	lipoma HMGIC fusion partner-like 1	Downward
	Lin7b	NM_011698.1	lin-7 homolog B (C. elegans)	Downward
	Mid1ip1	NM_001166635.1	Midl interacting protein 1 (gastrulation specific G12-like (zebrafish))	Downward
	Smco2	NM_027059.1	single-pass membrane protein with coiled-coil domains 2	Downward
	Tmem263	NM_001013028.2	transmembrane protein 263	Downward
	Tuba4a	NM_001313723.1	tubulin, alpha 4A	Downward
	Zscan4c	NM_001013765.2	zinc finger and SCAN domain containing 4C	Downward
	Zscan4d	NM_001100186.1	zinc finger and SCAN domain containing 4D	Downward
Unknown	0610040B10Rik	NR_027874.1	RIKEN cDNA 0610040B10 gene	lift
	1110007C09Rik	NM_026738.2	caspase recruitment domain family, member 19	lift
	1190002F15Rik	NR_037955.1	lncRNA downstream of Cdkn1b	lift
	1700124L16Rik	NR_105027.1	RIKEN cDNA 1700124L16 gene	lift
	1810063I02Rik	NR_130986.1	RIKEN cDNA 1810063I02 gene	lift
	2010204K13Rik	NR_027924.1	RIKEN cDNA 2010204K13 gene	lift
	2010320M18Rik	NR_029440.1	RIKEN cDNA 2010320M18 gene	lift
	2900076A07Rik	NR_045299.1	RIKEN cDNA 2900076A07 gene	lift
	4930444P10Rik	NM_001243238.2	RIKEN cDNA 4930444P10 gene	lift
	5830454E08Rik	NR_073359.1	RIKEN cDNA 5830454E08 gene	lift
	Armc12	NM_026290.3	armadillo repeat containing 12	lift
	C130036L24Rik	NR_015507.2	RIKEN cDNA C130036L24 gene	lift
	Ccdc59	NM_025602.3	coiled-coil domain containing 59	lift
	Commd8	NM_001356611.1	COMM domain containing 8	lift
	2010204K13Rik	NR_027924.1	RIKEN cDNA 2010204K13 gene	lift
	Gm12238	NR_028480.1	predicted gene 12238	lift
	Gm15008	NR_045917.1	predicted gene 15008	lift
	Gm16023	NR_040441.1	predicted gene 16023	lift
	H2-T9	NM_010399.2	histocompatibility 2, T region locus 9	lift
	I730030J21Rik	NR_045781.1	RIKEN cDNA I730030J21 gene	lift
	Kbtbd3	NM_001164574.1	kelch repeat and BTB (POZ) domain containing 3	lift
	LOC100504703	NR_040660.1	RIKEN cDNA A730063M14 gene	lift
	Mir8112	NR_106192.1	microRNA 8112	lift
	Mrpl52	NM_026851.2	mitochondrial ribosomal protein L52	lift
	Ndufc1	NM_025523.1	NADH: ubiquinone oxidoreductase subunit C1	lift
	Olfr649	NM_147055.1	olfactory receptor 649	lift
	R3hcc1	NM_001146012.2	R3H domain and coiled-coil containing 1	lift
	Snora33	NR_037680.1	small nucleolar RNA, H / ACA box 33	lift
	Snora70	NR_002899.1	small nucleolar RNA, H / ACA box 70	lift
	Snord15a	NR_002172.1	small nucleolar RNA, C / D box 15A	lift
	Snord15b	NR_002173.1	small nucleolar RNA, C / D box 14B	lift
	Zfp868	XM_011242291.2	zinc finger protein 868	lift
	1700061F12Rik	NR_038180.1	RIKEN cDNA 1700061F12 gene	Downward
	2700069I18Rik	NR_045905.2	RIKEN cDNA 2700069I18 gene	Downward
	4933430M04Rik	NR_045857.2	RIKEN cDNA 4933430M04 gene	Downward
	BB287469	NM_001177573.1	expressed sequence BB287469	Downward
	Eif3j1	NM_144545.4	eukaryotic translation initiation factor 3, subunit J1	Downward
	Gm11487	NM_001013393.1	predicted gene 11487	Downward
	Gm13078	NM_001085412.2	predicted gene 13078	Downward
	Gm13083	NM_001126324.1	predicted gene 13083	Downward
	Gm13152	NM_001039209.2	zinc finger protein 982	Downward
	Gm13242	NM_001103158.2	zinc finger protein 980	Downward
	Gm2016	NM_001122662.1	predicted gene 2016	Downward
	Gm2022	NM_001177574.1	predicted pseudogene 2022	Downward
	Gm21319	NM_001270722.1	predicted gene, 21319	Downward
	Gm4027	NM_001177564.1	predicted gene 4027	Downward
	Gm428	NM_001081644.1	predicted gene 428	Downward
	Gm5039	NR_003647.2	predicted gene 5039	Downward
	Gm5662	NM_001013824.3	predicted gene 5662	Downward
	Gm5868	NM_001024147.2	predicted gene 5868	Downward
	Gm8300	NM_001177565.1	predicted gene 8300	Downward
	Gm8764	NM_001270900.1	predicted gene 8764	Downward
	Gm8994	NM_001142734.1	predicted gene 8994	Downward
	Mir6340	NR_105758.1	microRNA 6340	Downward
	Nudt22	NM_026675.2	nudix (nucleoside diphosphate linked moiety X) -type motif 22	Downward
	Olfr1463	NM_001011840.1	olfactory receptor 1463	Downward
	Olfr376	NM_001172686.1	olfactory receptor 376	Downward
	Olfr450	NM_146445.1	olfactory receptor 450	Downward
	Olfr815	NM_146670.1	olfactory receptor 815	Downward
	Pramef6	NM_001085414.2	PRAME family member 6	Downward
	Rnase6	NM_001360117.1	ribonuclease, RNase A family, 6	Downward
	Snora52	NR_034049.2	small nucleolar RNA, H / ACA box 52	Downward
	Snora64	NR_002897.1	small nucleolar RNA, H / ACA box 64	Downward
	Snora74a	NR_002905.3	small nucleolar RNA, H / ACA box 74A	Downward
	Snord22	NR_004445.1	small nucleolar RNA, C / D box 22	Downward
	Tdpoz3	NM_207271.2	TD and POZ domain containing 3	Downward
	Tmem182	NM_001081198.1	transmembrane protein 182	Downward
	Usp17la	NM_007887.2	ubiquitin specific peptidase 17-like A	Downward
	Usp17lb	NM_001358922.1	ubiquitin specific peptidase 17-like B	Downward
	Usp17ld	NM_001001559.2	ubiquitin specific peptidase 17-like D	Downward
	Usp17le	NM_001256973.1	ubiquitin specific peptidase 17-like E	Downward
	Zscan4a	NR_033707.1	zinc finger and SCAN domain containing 4A	Downward

As a result, 175 genes were differentially expressed in fold change> 2 and FPKM> 5 in 2-cell embryos (Table 3). Specifically, 111 genes were up-regulated in the melatonin-treated group (SCNT-CROC + K + M) compared to the melatonin-untreated group (SCNT-CROC + K). In particular, the Amd1, Fam46C, Oxt, and Ppt2 involved in cell survival and tissue was up-regulated at the 2-cell embryo of melatonin treatment group (SCNT-CROC + K + M ). Also, Ctss , Dffa , Eif3f , Hacl1, Hspbp1 , Mob2 , Mrpl23 , Mrpl33 , Pmvk , Slc4a11 which are related to antioxidative function, inflammation and apoptosis And Wbscr22 was upgraded to a high level. On the other hand, cell death and regression-related genes ( Atp6v0c , Cd52 , Dapk2, Ddit4l , Duoxa2 , Nfkbia , Ptgds , Rdh12 And Rnd3 ) were down-regulated in 2-cell embryos of SCNT-CROC + K + M).

In blastocysts, 81 genes were differentially expressed in fold change> 2 and FPKM> 5 (Table S2). In particular, 20 genes were up-regulated in the melatonin-treated group (SCNT-CROC + K + M) compared to the melatonin-untreated group (SCNT-CROC + K). Amd1 , Fam46C , Oxt , and Ppt2 , which are associated with cell survival and tissue regeneration, were up-regulated in blastocysts of the melatonin-treated group. In particular, Gulo associated with antioxidant function, inflammation and apoptosis And Txnip were upregulated. On the other hand, genes associated with oxidative stress ( Adh1 , Car2 , Gsta3 , Gstm2 , Mb , Phlda2 , and S100a1 ) and cell death and regression-related genes ( Akr1c13 , Amd2 , Clu , Hist1h3a , Hspb1 , Il1rn , and Nat8 ) Treated group (SCNT-CROC + K + M). In addition, expression levels of cell and tumor proliferation related genes ( Efemp1 , Glipr1 , Lgals4 , Plac1 , Snora15 , Snora21 , Snora34 , and Anxa1 ) were decreased. In addition, some of the genes involved in the immune response ( Clec2f , Hrsp12 , and Plat ) were down regulated in the melatonin-treated group (SCNT-CROC + K + M) (Table 4).

Related features	Gene symbol	Gene ID	Gene name	control
Cell survival and tissue regeneration	Amd1	NM_009665.5	S-adenosylmethionine decarboxylase 1	lift
	Fam46C	NM_001142952.1	family with sequence similarity 46, member C	lift
	Oxt	NM_011025.4	oksytocin	lift
	Ppt2	NM_001302393.1	palmitoyl-protein thioesterase 2	lift
Antioxidant, inflammation and cell death	Gulo	NM_178747.3	gulonolactone (L-) oxidase	lift
	Txnip	NM_001009935.2	티오르 디오 톡 인	Downward
Oxidative stress	Adh1	NM_007409.2	alcohol dehydrogenase 1	Downward
	Car2	NM_001357334.1	carbonic anhydrase 2	Downward
	Gsta3	NM_001077353.2	glutathione S-transferase, alpha 3	Downward
	Gstm2	NM_008183.3	glutathione S-transferase, mu 2	Downward
	Mb	NM_001164047.1	myoglobin	Downward
	Phlda2	NM_009434.2	pleckstrin homology like domain, family A, member 2	Downward
	S100a1	NM_011309.3	S100 calcium binding protein A1	Downward
Cell death / regeneration	Akr1c13	NM_013778.2	aldo-keto reductase family 1, member C13	Downward
	Amd2	NM_007444.3	S-adenosylmethionine decarboxylase 2	Downward
	Clu	NM_013492.3	clusterin	Downward
	Hist1h3a	NM_013550.5	histone cluster 1, H3a	Downward
	Hspb1	NM_013560.2	heat shock protein 1	Downward
	Il1rn	NM_001039701.3	interleukin 1 receptor antagonist	Downward
	Nat8	NM_023455.3	N-acetyltransferase 8 (GCN5-related)	Downward
Cell and tumor proliferation	Efemp1	NM_146015.2	epidermal growth factor-containing fibulin-like extracellular metrix protein	Downward
	Glipr1	NM_028608.3	GLI pathogenesis-related 1 (glioma)	Downward
	Lgals4	NM_010706.2	lectin, galactose binding, soluble 4	Downward
	Plac1	NM_019538.4	placental specific protein 1	Downward
	Snora15	NR_003681.1	small nucleolar RNA, H / ACA box 15	Downward
	Snora21	NR_028078.1	small nucleolar RNA, H / ACA box 21	Downward
	Snora34	NR_034051.1	small nucleolar RNA, H / ACA box 34	Downward
	Anxa1	NM_010730.2	annexin A1	Downward
Immune response	Clec2f	NM_001277202.1	C-type lectin domain family 2, member f	Downward
	Hrsp12	NM_008287.3	reactive intermediate imine deaminase homolog	Downward
	Plat	NM_008872.3	plasminogen activator, tissue	Downward
	Snora75	NR_028478.1	small nucleolar RNA, H / ACA box 75	lift
Etc	Car4	NM_007607.2	carbonic anhydrase 4	Downward
	Hotairm1	NR_131181.1	Hoxa transcript antisense RNA, myeloid-specific 1	Downward
	Mt4	NM_008631.2	metallothionein 4	Downward
	Rhox6	NM_008955.1	reproductive homeobox 6	Downward
	Rmnd1	NM_025343.5	required for meiotic nuclear division 1 homolog	Downward
	S100a13	NM_009113.4	S100 calcium binding protein A13	Downward
	S100a16	NM_001356605.1	S100 calcium binding protein A16	Downward
	Serpinb6b	NM_011454.1	serine (or cysteine) peptidase inhibitor, clade B, member 6b	Downward
	Zscan4a	NR_033707.1	zinc finger and SCAN domain containing 4A	Downward
	Zscan4c	NM_001013765.2	zinc finger and SCAN domain containing 4C	Downward
	Zscan4d	NM_001100186.1	zinc finger and SCAN domain containing 4D	Downward
Unknown	Eif3j1	NM_144545.4	eukaryotic translation initiation factor 3, subunit J1	lift
	Gm19705	NR_045324.1	predicted gene, 19705	lift
	Gm5512	NR_002891.1	predicted gene 5512	lift
	Lmo4	NM_001161769.1	LIM domain only 4	lift
	Mir6340	NR_105758.1	microRNA 6340	lift
	Mir6363	NR_105782.1	microRNA 6363	lift
	Mir8094	NR_106169.1	microRNA 8094	lift
	Pisd-ps3	NR_003518.2	phosphatidylserine decarboxylase, pseudogene 3	lift
	Snora26	NR_031758.1	small nucleolar RNA, H / ACA box 26	lift
	Snora28	NR_033168.1	small nucleolar RNA, H / ACA box 28	lift
	Snora30	NR_034045.1	small nucleolar RNA, H / ACA box 30	lift
	Snora31	NR_028481.1	small nucleolar RNA, H / ACA box 31	lift
	Snora70	NR_002899.1	small nucleolar RNA, H / ACA box 70	lift
	4930461G14Rik	NR_040736.1	RIKEN cDNA 4930461G14 gene	Downward
	Ccdc160	NM_001034059.1	coiled-coil domain containing 160	Downward
	Ctsll3	NM_027344.3	cathepsin L-like 3	Downward
	Eif3j2	NM_001256055.1	eukaryotic translation initiation factor 3, subunit J2	Downward
	Gm5547	NR_045845.1	predicted gene 5547	Downward
	Gm5662	NM_001013824.3	predicted gene 5662	Downward
	Gm7334	NR_002700.1	predicted gene 7334	Downward
	Gm9992	NM_001142539.1	predicted gene 9992	Downward
	Hspb2	NM_001164708.1	heat shock protein 2	Downward
	Klra22	NM_053152.2	killer cell lectin-like receptor subfamily, member 22	Downward
	LOC100504703	NR_040660.1	RIKEN cDNA A730063M14 gene	Downward
	Mir1191	NR_035422.1	microRNA 1191	Downward
Unknown	Mir6236	NR_105744.1	microRNA 6236	Downward
	Mir6516	NR_105853.1	microRNA 6516	Downward
	Ms4a10	NM_023529.2	membrane-spanning 4-domains, subfamily, member 10	Downward
	Pinlyp	NM_001037143.2	phospholipase A2 inhibitor and LY6 / PLAUR domain	Downward
	Pisd-ps1	NR_003517.1	phosphatidylserine decarboxylase, pseudogene 1	Downward
	Platr21	NR_045455.1	pluripotency associated transcript 21	Downward
	Pramef25	NM_001126315.2	PRAME family member 25	Downward
	Snora47	NR_034043.1	small nucleolar RNA, H / ACA box 47	Downward
	Snora64	NR_002897.1	small nucleolar RNA, H / ACA box 64	Downward
	Snora81	NR_034048.1	small nucleolar RNA, H / ACA box 81	Downward
	Tmem40	NM_001168256.1	transmembrane protein 40	Downward
	Usp17lb	NM_201409.2	ubiquitin specific peptidase 17-like B	Downward

Example  6. Embryonic transfer and implantation monitoring

The reconstituted embryos cultured in the blastocyst stage under melatonin-treated or non-treated conditions (SCNT-CROC + K and SCNT-CROC + K + M group) were suspended in a 2.5 day pseudo- pregnant) female ICR mice. At this time, SCNT-CROC + K group and SCNT-CROC + K + M group were transplanted to the left and right uterus of the same mouse, respectively. The embryo transfer rate in female mice was confirmed at 7.5 days after the sexual intercourse of the mice. After the mice were euthanized, the normal uterus was washed with saline and resected and the adhered fats were removed. The fat removed tissue was photographed to visualize the implantation (Fig. 6a, Fig. 6b).

As a result, the transplantation rate was significantly increased in the melatonin-treated group (SCNT-CROC + K + M) compared with the melatonin untreated group (SCNT-CROC + K) (66.2% (51/77) vs. 42.9% 33/77), p < 0.05; Fig. 6c).

Example 7: Derivation of mouse embryonic stem cells from SCNT blastocysts

To obtain outgrowths, the hatched blastocysts obtained in two groups (SCNT-CROC + K and SCNT-CROC + K + M) were injected into mitotic inactivated mouse embryonic fibroblasts of mouse embryonic stem cell (mESC) MEF) feeder cells. (Gibco / Invitrogen, Grand Island, NY) and 1.5 x 10 &lt; ³ &gt; cells per well were incubated with 20% KRS, 0.1 mM beta-mercaptoethanol, 1% nonessential amino acids, 100 units / ml penicillin, units / mL DMWM / F12 containing recombinant mLIF (Chemicon, Temecula, CA) was used as the culture medium for mES cell culture. First, the debris was mechanically transferred to the MEF feeder cells and then transferred using trypsin-EDTA. All established mECS lines were monitored and characterized by morphology and alkaline phosphatase staining. The alkaline phosphatase activity was evaluated by histochemical staining. The colonies were fixed with 4% paraformaldehyde at room temperature for 1 min and washed twice with PBS. The alkaline phosphatase substrate solution (10 ml FRV-alkaline solution, 10 ml naphthol AS-BI alkaline solution, alkaline phosphatase kit, Sigma-Aldrich) At room temperature for 30 minutes. The alkaline phosphatase activity was detected by a colorimeter using an optical microscope (Fig. 7A).

As a result, the induction rate of mESC was significantly higher in the melatonin-treated group than in the untreated group (21.3% (10/47) vs. 5.6% (2/36), p <0.05;

Example 8. Statistical analysis

All experiments were repeated at least 3 times. Results were expressed as mean or mean ± standard error of mean. Embryonic development was analyzed by one-way analysis of variance (ANOVA) by DUNCAN using SAS software and transplantation. ESC-derivation rates were analyzed by Chi-square test and p <0.05 was considered statistically significant.

Claims (21)

1. A composition for increasing the efficiency of embryo development or the formation of an embryo, including melatonin.
2. The composition of claim 1, wherein the melatonin is included at a concentration of between 5 μM and 20 μM.
3. The composition of claim 1, further comprising an agent that reduces H3K9me3 methylation.
4. 4. The composition of claim 3 wherein the agent that reduces H3K9me3 methylation is an agent that increases the expression of a member of the KDM4 family of histone demethylating enzymes.
5. 5. The composition according to claim 4, wherein the agent increases the expression or activity of KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD2D) or a combination thereof.
6. The composition according to claim 1, wherein the melatonin reduces active oxygen and / or apoptosis of the embryo.
7. The composition according to claim 1, wherein the melatonin reduces the cell death of the embryo.
8. The composition of claim 1, wherein the embryo development is development of an embryo into a blastocyst or formation of a blastocyst.
9. 2. The composition of claim 1, wherein the embryo is formed through in vitro fertilization.
10. The composition according to claim 1, wherein the embryo is formed by implanting nuclei of somatic cells in an oocyte removed from the nucleus.
11. A method of increasing the efficiency of embryonic development or the formation of an embryo, comprising culturing the embryo in a medium containing melatonin.
12. 13. The method of claim 11, comprising contacting the oocyte with an agent that reduces H3K9me3 methylation.
13. 12. The method according to claim 11, wherein the egg is frozen and then melted.
14. 12. The method of claim 11, wherein when the embryo is in a 2- celled state, Amd1 , Fam46C , Oxt, Ppt2 , Ctss , Dffa , Eif3f , Hacl1 , Hspbp1 , Mob2 , Mrpl23 , Mrpl33 , Pmvk , Slc4a11 , Lt ; RTI ID = 0.0 &gt; Wbscr22 &lt; / RTI &gt;
15. 12. The method of claim 11, wherein when the embryo is in a 2- celled state, Atp6v0c , Cd52 , Dapk2, Ddit4l , Duoxa2 , Nfkbia , Ptgds , Rdh12 And Rnd3 in the expression of at least one gene.
16. 12. The method of claim 11, wherein when the embryo develops into a blastocyst, Amd1 , Fam46C , Oxt , Ppt2 , Gulo And Txnip are increased.
17. 12. The method of claim 11, wherein when the embryo develops into a blastocyst, it is selected from the group consisting of Adh1 , Car2 , Gsta3 , Gstm2 , Mb , Phlda2 , S100a1 , Akr1c13 , Amd2 , Clu , Hist1h3a , Hspb1 , Il1rn , Nat8, Efemp1 , Glipr1 , Lgals4 , Plac1 , the method of Snora15, Snora 21, Snora 34, Anxa1, Clec2f, Hrsp12, Plat and that any one or more genes selected from the group consisting of reduction.
18. 12. The method according to claim 11, further comprising developing the embryo obtained in the culturing step as an individual.
19. An embryo produced by the method of claim 11.
20. A blastocyst produced by the method of claim 11.
21. 12. An embryonic stem cell prepared by the method of claim 11.

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