KR20170082856A - Method for enhancing of reprogramming of cloned embryos utilizing Scriptaid - Google Patents
Method for enhancing of reprogramming of cloned embryos utilizing Scriptaid Download PDFInfo
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- KR20170082856A KR20170082856A KR1020160002125A KR20160002125A KR20170082856A KR 20170082856 A KR20170082856 A KR 20170082856A KR 1020160002125 A KR1020160002125 A KR 1020160002125A KR 20160002125 A KR20160002125 A KR 20160002125A KR 20170082856 A KR20170082856 A KR 20170082856A
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Abstract
The present invention relates to a method for reprogramming a cloned embryo using Scriptaid, and more particularly, to a method for improving reprogramming of a cloned embryo using Scriptaid, lt; RTI ID = 0.0 > miRNA) -152 < / RTI > to improve reprogramming of the cloned embryo.
The present invention has the effect that Scriptaid inhibits Dnmt1 expression in a porcine cloned embryo and increases the expression of miR-152, thereby improving the development of a cloned embryo to a pre-implantation stage.
Description
The present invention relates to a method for reprogramming a cloned embryo using Scriptaid, and more particularly, to a method for improving reprogramming of a cloned embryo using Scriptaid, lt; RTI ID = 0.0 > miRNA) -152 < / RTI > to improve reprogramming of the cloned embryo.
Reprogramming is the process by which epigenetic markers, such as DNA methylation, are altered during mammalian development. After fertilization, some cells constituting the newly formed embryo move along the germinal redge, and eventually become germ cells such as sperm and egg. By genomic imprinting, parents' genomes are represented differently, but all are reprogrammed during the process of becoming germ cells. Therefore, during gametogenesis c, primordial germ cells are re-established according to the gender of the embryo after clearing the DNA methylation pattern originally possessed by the parents.
After the sperm and egg are modified, the parental genome is once again demethylated and remethylated. This reprogramming causes the newly formed embryo to have totipotency, It is the process of erasing the characteristic of the image. In vitro manipulation of pre-implantation embryos, such as somatic cell nuclear transfer (SCNT), has been shown to cause abnormalities in the methylation pattern of the imprinted genomic locus, which has been shown to have a devastating effect on replicated animals . Reprogramming can be artificially induced by overexpressing stem cell-associated genes exogenously as well as nuclear replacement of somatic cells.
Abnormal epigenetic reprogramming in mammalian somatic cell nuclear transfer (SCNT) embryos, a cloned embryo, is responsible for low replication efficiency. Scriptaid acts to inhibit Class I and Class II a / b histone deacetylase (HDAC). Scriptaid is a hydroxamic acid class of histone deacetylase inhibitor (HDACi) that is less toxic than other HDACi, and increases histone acetylation as a whole, inducing strong transcriptional activity and protein expression. In addition, microRNA (microRNA, miRNA) regulates HDAC and histone acetylation, and HDAC also plays a role in regulating miRNA expression. Thus, in view of this, the inventors have studied how to improve the reprogramming of cloned embryos by processing Scriptaid.
It is an object of the present invention to provide a method for improving reprogramming of a mammalian embryo embryo by treating Scriptaid.
Another object of the present invention is to provide a medium composition for reprogramming a mammalian cloned embryo comprising Scriptaid.
In order to accomplish the object of the present invention, the present invention provides a method for producing a recombinant embryo comprising: (a) preparing a cloned embryo of a mammal other than a human; And (b) culturing the cloned embryo in a medium that has been treated with Scriptaid.
In one embodiment of the present invention, in step (b), the Scriptaid may be treated at a concentration of 100 to 300 nM, but is not limited thereto.
In one embodiment of the present invention, in the step (b), the Scriptaid processing may be performed for 10 to 20 hours, but is not limited thereto.
In one embodiment of the present invention, in step (a), the mammal other than the human may be a pig, a sheep, a dog, a horse, or a goat, but is not limited thereto.
In one embodiment of the invention, the reprogramming enhancing method of a mammalian cloned embryo of the present invention can improve the in vitro development rate of a somatic cell cloned embryo or increase the number of blastocyst cells.
In one embodiment of the present invention, the method of reprogramming a mammalian cloned embryo of the present invention inhibits the expression of DNA methyltransferase 1 (Dnmt1) or the expression of microRNA (miRNA) -152 .
Further, in order to achieve the object of the present invention, the present invention provides a culture composition comprising Scriptaid, which provides a medium composition for reprogramming a mammalian cloned embryo except for a human.
In one embodiment of the present invention, the culture medium may be treated with the Scriptaid at a concentration of 100 to 300 nM, but is not limited thereto.
In one embodiment of the present invention, the Scriptaid treatment may be performed for 10 to 20 hours, but the present invention is not limited thereto.
The present invention has the effect that Scriptaid inhibits Dnmt1 expression in a porcine cloned embryo and increases the expression of miR-152, thereby improving the development of a cloned embryo to a pre-implantation stage.
FIG. 1 (a) is a graph showing the percentage of blastocysts according to the Scriptaid concentration,
FIG. 1 (b) is a graph showing cell numbers of blastocysts according to Scriptaid treatment time,
1 (c) is a graph showing the percentage of blastocysts according to the Scriptaid concentration,
FIG. 1 (d) is a graph showing cell numbers of blastocysts according to Scriptaid treatment time.
FIG. 2 is a graph showing the developmental stages of the in vitro maturation embryo according to the Scriptaid treatment.
FIG. 3 is a graph showing changes in the amount of DNA methyltransfarase (Dnmt) expressed in a somatic cell cloned embryo treated with Scriptaid at a concentration of 300 nM for 20 hours.
4 is a graph showing changes in the amount of micro RNA (miR) expression in a somatic cell cloned embryo treated with Scriptaid at a concentration of 300 nM for 20 hours.
5 is a graph showing the development rate of an in vitro maturation embryo at the time of Scriptaid alone treatment and mixed treatment with Scriptaid and TSA.
Hereinafter, a method for improving reprogramming of a mammalian cloned embryo and a medium composition for reprogramming a mammalian cloned embryo according to an embodiment of the present invention will be described in detail.
The present invention relates to a method for improving reprogramming of a mammalian embryo embryo, excluding a human, comprising the steps of:
(a) preparing a cloned embryo of a mammal other than a human; And
(b) culturing the cloned embryo in a Scriptaid-treated medium.
The present invention also relates to a culture composition comprising Scriptaid, which relates to a medium composition for improving reprogramming of a mammalian cloned embryo.
In the present invention, the mammalian cloned embryo is preferably a mammalian cloned embryo except human. The mammals other than humans are preferably cows, pigs, sheep, including pigs, sheep, dogs, cows, horses, chlorine, etc., and most preferably pigs.
The medium in the present invention preferably uses a basic medium used for culturing mammalian oocytes. The basic medium varies depending on the species of the mammal, but includes any one or more selected from the group consisting of inorganic salts, carbon sources, amino acids, bovine serum albumin, and coadjuvants, and includes 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. The amino acids include essential amino acids such as glutamine, Other auxiliaries may be other trace elements and buffers. The basal medium used for the in vitro maturation culture may further comprise antibiotics.
The concentration of Scriptaid contained in the medium of the present invention is not particularly limited and a person skilled in the art can select and determine an appropriate concentration for improving reprogramming of a mammalian embryo embryo. The concentration of Scriptaid contained in the medium is preferably 5 to 600 nM, more preferably 50 to 300 nM, even more preferably 100 to 300 nM, most preferably 100 nM. In the present invention, the reprogramming-enhancing effect of the mammalian embryo embryo begins to manifest itself above a concentration of 100 nM.
The time for treating Scriptaid in the medium of the present invention is not particularly limited, and a person skilled in the art can determine an appropriate treatment time for improving the reprogramming of the mammalian embryo embryo. Preferably, the time for treating Scriptaid in the medium is 1 to 40 hours, more preferably 10 to 20 hours.
Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited to the following examples.
[ Example 1] Obtaining eggs
At the local slaughterhouse, I got pig ovaries before adolescence. Cysteine, 10 ng / mL epidermal growth factor (EGF), 10 IU / mL PMSG (pregnant mare ' s serum gonadotropin) and 10 IU / mL hCG (GIBCO BRL, Grand Island, NY, USA) supplemented with human chorionic gonadotropin (TCM) at 38.5 ° C and 5% CO2 (v / v) under mild mineral oil.
[Example 2] Developmental rate of cloned embryos according to the concentration of Scriptaid
In order to confirm the development rate of the cloned embryos to the blastocysts and the cell number of the blastocysts according to the concentration of Scriptaid, the cells were cultured in a culture medium of 100 nM, 300 nM and 500 nM of Scriptaid in the culture medium of the pig embryo embryo, , And the development rate of blastocysts and the number of blastocyst cells were examined on the 7th day after culturing. The results are shown in FIGS. 1A and 1B, respectively.
As shown in FIGS. 1A and 1B, in the 300 nM, 500 nM Scriptaid-treated group, the developmental rate to the blastocyst was significantly increased and the number of the blastocyst cells was significantly increased compared to the 100 nM treated group and the control group.
In addition, in order to confirm the development rate of the cloned embryos to the blastocysts and the cell number of the blastocysts according to the processing time of Scriptaid, the culture medium of the pig embryo embryos was treated with 300 nM of Scriptaid and cultured for 10 hours and 20 hours. And cultured in vitro by replacing with fresh culture medium. The development rate of blastocysts and the number of blastocyst cells were examined on the 7th day after culturing, and the results are shown in FIG. 1C and FIG. 1D, respectively.
1C and FIG. 1D, the developmental rate to the blastocyst was significantly increased in the 20 hour treatment group compared to the control group, and the number of blastocyst cells was significantly increased in the 10 hour treatment group and the 20 hour treatment group as compared with the control group can confirm.
[Example 3] Investigation of developmental stage of in vitro maturation embryo by Scriptaid treatment
In order to confirm the developmental stage of the in vitro maturation embryos according to the Scriptaid treatment, Scriptaid treatment groups of 100 nM, 300 nM and 500 nM in the control group were cultured in in vitro culture medium, and on the 1st, 2nd, 4th and 7th days, And the developmental rate to blastocysts and blastocysts were examined. The results are shown in Fig.
2, there was no significant difference between the Scriptaid treatment group and the control group in the 2-cell and 4-cell treatment groups, but the development rate of blastocysts and blastocysts was significantly increased in the Scriptaid-treated group compared to the control group.
[Example 4] Examination of gene expression and microRNA expression by Scriptaid treatment
In order to confirm gene expression and microRNA expression according to the Scriptaid treatment, the culture medium of the pig pig embryo embryo was treated with 300 nM of Scriptaid for 20 hours, then the Scriptaid was removed and replaced with a fresh culture medium for in vitro culture, On day 7, expression of miR-29b, miR-148a and miR-152 in Dnmt1, Dnmt3a, Dnmt3b and microRNA (miRNA, miRNA) in DNA methyltransferase (Dnmt) was examined. And FIG. 4, respectively.
As shown in FIG. 3, the expression of Dnmt1 was significantly inhibited by the treatment with Scriptaid. As shown in FIG. 4, the expression of miR-152 significantly increased in the case of treatment with Scriptaid. In other words, Scriptaid confirmed that Dnmt1 and miR-152 could be regulated to improve embryo development to the pre-implantation stage.
[Example 5] Investigation of the development rate of Scriptaid alone treatment and mixing treatment with TSA
In order to determine the effect of Scriptaid alone on the developmental rate of the cloned embryos, the nucleated embryos were divided into a single treatment group of 300 nM Scriptaid, a mixed treatment group of 300 nM Scriptaid and 50 nM TSA (Trichostatin A) for 20 hours After removing the Scriptaid and replacing it with fresh culture medium, the in vitro culture was carried out. The developmental rate of the blastocyst was examined on the 7th day of culture, and the result is shown in FIG. It is known as a substance that synchronizes the cell cycle of immature oocytes used for the production of transgenic animals or somatic cell clones using TSA (see Korean Patent Publication No. 2015-0081462).
5, in the group treated with 300 nM Scriptaid alone and in the group treated with 300 nM Scriptaid and 50 nM TSA, the development rate to the blastocyst was significantly higher than that in the control group. However, in the group treated with 300 nM Scriptaid alone and the mixture treatment with 300 nM Scriptaid and 50 nM TSA There was no significant difference between the groups.
That is, therefore, it can be seen that the single treatment of 300 nM Scriptaid is the same effect as the mixing treatment with TSA, and it is confirmed that the effect on the replication embryo development rate of Scriptaid is remarkably superior to that of known materials.
Claims (9)
(a) preparing a cloned embryo of a mammal other than a human; And
(b) culturing the cloned embryo in a medium treated with Scriptaid.
Wherein in step (b), the scriptite is treated at a concentration of 100 to 300 nM.
Wherein in step (b), the Scriptaid is treated for 10 to 20 hours.
Wherein the mammal other than the human in step (a) is a pig, a sheep, a dog, a horse, or a goat.
Wherein said method enhances the in vitro development rate of a somatic cell cloned embryo or increases the number of blastocyst cells.
Wherein said method inhibits the expression of DNA methyltransferase 1 (Dnmt1) or increases the expression of miR-152.
A medium composition for improving reprogramming of a mammalian cloned embryo other than a human.
Wherein the Scriptid is treated at a concentration of 100 to 300 nM.
Wherein the Scriptaid is treated for 10 to 20 hours.
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| KR1020160002125A KR20170082856A (en) | 2016-01-07 | 2016-01-07 | Method for enhancing of reprogramming of cloned embryos utilizing Scriptaid |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20220079747A (en) * | 2020-12-04 | 2022-06-14 | 대한민국(농촌진흥청장) | Method for enhancing developmental rate of porcine somatic cell nuclear transfer embryos using microRNA-148a direct injection |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20220079747A (en) * | 2020-12-04 | 2022-06-14 | 대한민국(농촌진흥청장) | Method for enhancing developmental rate of porcine somatic cell nuclear transfer embryos using microRNA-148a direct injection |
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