KR102520268B1 - Method for enhancing developmental rate of porcine somatic cell nuclear transfer embryos using microRNA-148a direct injection - Google Patents

Abstract

The present invention relates to a method for improving the development rate of porcine nuclear transfer eggs through direct injection of miRNA-148a, and shows that the cleavage rate is increased at the very beginning of culture by microinjecting miRNA-148a mimics into pig nuclear transfer cloned eggs. By confirming, it can be usefully used to improve the pig nuclear transfer egg development rate.

Description

Method for enhancing developmental rate of porcine somatic cell nuclear transfer embryos using microRNA-148a direct injection}

The present invention relates to a method for improving the development rate of porcine somatic cell nuclear transfer eggs through direct injection of microRNA-148a.

microRNAs (or miRNAs) are small non-coding RNAs that suppress gene expression at the post-transcriptional regulatory level. microRNA consists of 18 to 25 nucleotides on average and forms a hairpin structure (Ambros, Nature , 431, 7006:350-5 (2004); Bartel, Cell , 116, 2:281-97 (2004)) . By complementarily binding to the 3'UTR region of the target gene sequence, degradation of mRNA or translation into protein is suppressed, and about 5000 or more human genes have been identified as targets of microRNA (Lee et al., Cell , 75, 5). :843-54, (1993)). The functions of microRNAs in vivo are diverse, including cell differentiation, proliferation, development, regulation of metabolism, angiogenesis, and apoptosis (Friedman et al., Genome Res ., 19, 1:92-105 (2009); Krek et al. ., Nature Getenics , 37, 5:495-500 (2005)).

microRNA-148a regulates low-density lipoprotein metabolism by inhibiting renin receptors, or by confirming that microRNA-148a is silenced while DNA methyltransferase 1 (DNMT1) is overexpressed in gastric cancer, associations with gastric cancer have also been reported. (Wang et al., PLOS ONE , 15, 5 (2020); Zhu et al., Med Oncol . 29, 4 (2012)).

On the other hand, somatic cell nuclear transfer (SCNT) is a technique for transplanting or replacing the nucleus of a somatic cell in an egg whose nucleus has been removed. It is possible to clone an animal having the same genetic information using somatic cells of a specific individual. . Further, direct injection (or microinjection) refers to injecting a substance into an organ, tissue, cell, or intracellular organelle of a living organism. Such substances include chemicals, polynucleotides, or polypeptides. The direct injection includes germ cell microinjection, zygote microinjection, embryo microinjection and somatic cell microinjection.

However, there are cases in which the development rate of somatic cloned eggs is reduced because reprogramming is not normally performed. In particular, in the case of pigs, the rate of in vitro development of nuclear-transferred eggs until the blastocyst stage is very low, less than 5%, and the number of cells is small, and it is difficult to achieve continuous development of nuclear-transferred eggs (Du et al., Theriogenology , 282 , 2095 (1999); Tao et al., Cloning , 1, 55 (1999)).

Reprogramming refers to the process by which epigenetic marks, such as DNA methylation, are changed during mammalian development. It is important to increase the initial development rate of nuclear-transferred eggs only when reprogramming occurs well to start cleavage and proceed with embryonic development.

Recently, it has been reported that microRNAs can be used to regulate the expression of early developmental factors as well as reprogramming. Direct inhibition of mRNA or indirect inhibition of up-stream factors in the signaling pathway can positively or negatively affect the development of fertilized eggs. Also, a single miRNA can affect multiple mRNAs simultaneously.

Accordingly, the present inventors conducted intensive research to develop a method for effectively increasing the development rate of porcine nuclear transferred eggs, and as a result, it was found that direct injection of a miRNA-148a mimic into swine nuclear transferred eggs increases the cleavage rate in the early development of porcine nuclear transferred eggs. Confirmation completed the present invention.

An object of the present invention is to provide a method for improving the development rate of nuclear-transferred eggs, comprising the step of directly injecting microRNA-148a into porcine nuclear-transferred eggs in order to provide an excellent effect with high cleavage at the very beginning of culture.

In order to achieve the above object, the present invention provides a method for improving the development rate of nuclear-transferred eggs, comprising the step of directly injecting microRNA-148a into nuclear-transferred eggs.

In addition, the present invention is 1) removing the nucleus from the mature egg

2) Preparing donor cells

3) Fusing the cytoplasm and nucleus of the donor cell by electrical stimulation twice

4) Provided is a method for improving the development rate of nuclear-transferred eggs, comprising the step of directly injecting microRNA-148a into the nuclear-transferred eggs obtained in step 3).

When the microRNA-148a mimic of the present invention is microinjected into cloned pig somatic cells, it shows an excellent cleavage rate on the first day of culture, which is the early stage of development. 148a Direct injection can improve the development rate of pig nuclear-transferred eggs.

1 is a diagram showing the cleavage rate as a result of microinjection of a miRNA-148a mimic, a microRNA-148a inhibitor, and a control into pig nuclear transfer cloned eggs.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for improving the development rate of nuclear-transferred eggs, comprising the step of directly injecting microRNA-148a into nuclear-transferred eggs.

As the nuclear transfer egg, matured eggs collected through in vitro maturation (IVM) for 30 to 70 hours may be used, but are not limited thereto.

The egg may be collected from a mammal other than human, and the mammal may be a pig, but is not limited thereto.

The nuclear transfer means removing a nucleus from an egg and then introducing a donor nucleus obtained from another cell into the egg.

The somatic cell nuclear transfer refers to nuclear transfer when the cell including the donor nucleus is a somatic cell, and through this, it is possible to clone an animal having the same genetic information using a somatic cell of a specific individual.

The microRNA-148a regulates low-density lipoprotein metabolism by inhibiting the renin receptor, or by confirming that the microRNA-148a is silenced while DNA methyltransferase 1 (DNMT1) is overexpressed in gastric cancer, the association with gastric cancer, etc. It is a therapeutic target for a variety of cancers, including In addition, it has a mechanism to promote DNA hypomethylation by inhibiting DNMT1.

The direct injection is also referred to as microinjection, and means injecting a substance into an organ, tissue, cell, or intracellular organelle of a living organism. The material may include a chemical substance, polynucleotide, or polypeptide, and more specifically, may be microRNA-148a. The direct injection may include germ cell microinjection, zygote microinjection, embryo microinjection and somatic cell microinjection.

The germ cell microinjection refers to microinjection of a material containing a polynucleotide into germ cells, and may include a technique of obtaining a transgenic animal through a fertilization step, a differentiation step, and the like after microinjection. The zygote microinjection means microinjection of a material containing a polynucleotide into the zygote, and may include a technique of obtaining a transgenic animal through a differentiation step after microinjection. The embryo microinjection refers to microinjection of a polynucleotide-containing material into the embryo, and may include a technique of obtaining a transgenic animal through a differentiation step after microinjection. The somatic cell microinjection refers to microinjection of a material including a polynucleotide into somatic cells.

In a specific embodiment of the present invention, a conjugate microinjection method may be used, but is not limited thereto. The zygote microinjection method has the advantage of being able to confirm the effect of microRNA on the early stage of embryonic development, such as the initial cleavage rate, by treating the zygote rather than the microinjection method of germ cells or microRNA treatment of somatic cells. Since it is not known how long the half-life is depending on the type of microRNA, when microRNA is injected by germ cell microinjection or somatic cell microinjection, even if the initial cleavage rate increases, whether it is a direct effect of the microRNA or an indirect effect due to a chain reaction is unknown. There is an unknown problem. Accordingly, the present inventors present data that the effect of microRNA-148a by injecting microRNA into the zygote has an active time (half-life) of about 1 day, and that the effect of microRNA is more intuitive. In addition, this method can provide a more convenient and stable experimental method than germ cell microinjection or somatic cell microinjection. Since germ cells (sperm) and somatic cells are smaller in size than zygotes, direct injection requires advanced technology and equipment. There is no choice but to have a large error in injecting only the constant. Accordingly, in the present invention, the direct injection method of the conjugate may be used.

In the present specification, the direct injection and microinjection may be used interchangeably.

Direct injection of the miRNA-148a can improve the development rate of nuclear-transferred eggs by increasing the initial cleavage rate of porcine somatic cell nuclear-transferred eggs.

The initial period means the first to third days of culture after direct injection of miRNA-148a, and more specifically, it was confirmed that there was a significant effect on the increase in cleavage rate on the first day of culture.

In addition, the present invention is 1) removing the nucleus from the mature egg

2) Preparing donor cells

3) Fusing the cytoplasm and nucleus of the donor cell by electrical stimulation twice

4) Provided is a method for improving the development rate of nuclear-transferred eggs, comprising the step of directly injecting microRNA-148a into the nuclear-transferred eggs obtained in step 3).

In step 1), the mature oocyte may be a pig oocyte collected through in vitro maturation for 30 to 70 hours, more specifically, an oocyte collected through in vitro maturation for 40 to 50 hours, but is not limited thereto.

In step 1), after treatment with hyaluronidase, the collected oocytes are pipetted in PBS or a TCM199 culture medium-based micromanipulation culture medium to remove cumulus cells.

In a specific embodiment, the hyaluronidase can be treated for 1 to 5 minutes, more specifically 1 to 3 minutes, and pipetting with a micromanipulation culture medium is 20 to 60 times, more specifically 30 to 50 times It can be performed twice, but is not limited thereto.

The micromanipulation culture medium contained 0.001 to 0.01% HEPES, 0.01 to 0.5% BSA, 0.001 to 0.005% penicillin G, and 0.001 to 0.006% streptomycin in TCM199. It may be prepared by adding, but is not limited thereto.

In the step 1), transferring the oocytes from which the cumulus cells have been removed to a culture medium for NT drop containing 0.0003 to 0.001% cytochalasin B in the micromanipulation medium and selecting the step.

The selected oocytes may include metaphase II (MII) stage oocytes.

In step 1), the egg is stained with Hoechst 33342 and then the nucleus is extracted using a micromanipulation system.

The Hoechst 33342 is a DNA dye and structurally binds to the poly AT rich region of the minor groove of DNA.

The micromanipulation system is attached to a microscope and used together with an injector to inject specific substances (DNA, RNA, protein, cells, etc.) according to the purpose of floating cells, and is used for nuclear transfer and embryonic stem cell transplantation. It consists of a manipulator, a microinjector and a microscope. It is an equipment that enhances the reproducibility and accuracy of experiments with automatic coordinate recognition and positioning.

The egg may be collected from a mammal other than human, and the mammal may be a pig, but is not limited thereto.

In step 2), the donor cells may be pig somatic cells, and more specifically, pig ear-derived fibroblasts, but is not limited thereto.

In step 3), electrical stimulation is performed in a fusion solution (0.28 to 0.3 M mannitol, 0.1 mM MgSO ₄ 7H ₂ 0, 0,05 mM CaCl ₂ 2H ₂ O, 0.01% BSA or 0.1% PVA). The egg is placed, and the voltage is 100 to 200 V, the length is 0.01 to 0.1 ms, and the pulse width is 30 to 70 μs. More specifically, the voltage is 130 to 160 V, the length is 0.02 to 0.04 ms. , The pulse width may be performed under the condition of 40 to 60 μs, but is not limited thereto.

The two electrical stimulations may be performed within the same range.

The 'nuclear transfer', 'microRNA-148a', and 'direct injection' are as described above.

In the present invention, when microRNA-148a mimic was microinjected into porcine somatic cell nuclear transfer eggs, an excellent cleavage rate was confirmed on the first day of culture, which is an early stage of development.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples.

However, the Examples and Experimental Examples to be described below are only to specifically illustrate the present invention in one aspect, but the present invention is not limited thereto.

<Example 1>

1-1. Removal of nuclei from mature pig eggs

Mature pig eggs were collected through in vitro maturation (IVM) for about 44 hours. The Cumulus cells of the collected oocytes were treated with hyaluronidase for 2 minutes, and then micromanipulation culture medium was used for 30 to 30 minutes at an appropriate speed so that the oocytes did not explode with a pipette. It was removed by pipetting about 50 times.

After transferring the oocytes from which cumulus cells have been removed to the micromanipulation culture medium, find and select only the oocytes with polar bodies, transfer them to a 4-well dish containing the micromanipulation culture medium, and place them in an incubator. put in

Selected metaphase II (MII) oocytes were stained with 10 μg/mL Hoechst 33342 (Thermo) at 38.5° C. for 5 minutes. The stained oocytes were transferred to a micromanipulation culture medium containing 5 μg/mL cytochalasin B, and then the nucleus was extracted using a micromanipulation system.

1-2. Preparation of donor cells

Pig ear-derived fibroblast cells were used as donor cells for somatic cell nuclear transfer. The frozen donor cells were prepared by washing three times with a cell washing culture medium after dissolution.

1-3. Fusion of cytoplasm and donor cell by two times of electrical stimulation

The donor cells prepared in steps 1 and 2 are injected into the perivitelline space of the oocyte from which the nucleus was extracted. For the fusion of the cytoplasm and the donor cells, a chamber is installed on a warm plate, 100 μl of Fusion culture medium is added, the pedal is stepped on 5 times, and then removed and filled with about 60 μl. The fusion medium contains 0.28 to 0.3 M mannitol, 0.1 mM MgSO ₄ 7H ₂ 0, 0,05 mM CaCl ₂ 2H ₂ O, 0.01% BSA or 0.1% PVA, and has a pH of 7.0-7.4.

Thereafter, electrical stimulation was given twice under the conditions shown in Table 1 below. Oocytes were transferred from a micromanipulated 4-well dish to a 4-well dish in which the micromanipulated medium and the fusion medium were mixed at ratios of 100:300, 200:200, 300:100, and 0:400. After transferring the eggs sequentially from well 1, they were placed in the chamber. When the number of oocytes was large, the process was performed in divided rather than at once. The number of eggs working in the chamber was about 10 to 15.

After adjusting the polar body to be level with the chamber, electrical stimulation was applied. Oocytes activated by electrical stimulation were placed in an incubator for about 15 minutes to induce fusion.

First and second electrical stimulation conditions condition unit Voltage Voltage 147 V Battlefield Interval 0.03 ms pulse width Pulse width 50 ㎲

1-3. Microinjection of microRNA

For the preparation of microRNA, the microRNA stock stored at -20 ° C was put on ice and dissolved. 10 μl of the dissolved microRNA stock was injected into an ICSI microglass pipette (ORIGIO, MIC-SIM-30). MicroRNA was microinjected into the cytoplasm of activated oocytes using a micromanipulation system.

At this time, it was injected briefly while paying attention to the pressure so as not to burst the cytoplasm. As a control, RNA-free water was injected in the same way. The microinjected oocytes were placed in PZM-3 culture medium, and then the oocytes were washed. Then, it was transferred to 4 wells and placed in an O ₂ incubator. Embryos were observed for 3 days, cleavage was confirmed and recorded, and the culture medium was replaced at 2-day intervals.

<Experimental Example 1> Evaluation of cleavage on the first day of culture, which is the initial stage of miRNA-148a microinjection

A total of three or more repetitions (four times) of the experiment were performed, and as a result of microinjection of miRNA-148a mimic into pig nuclear transfer cloned eggs, the cleavage rate was higher until the first day of culture compared to the control group injected only with RNA free water. confirmed that On the other hand, when the miRNA-148a inhibitor was injected, it was confirmed that the cleavage rate decreased until the first day of culture compared to the control group injected only with RNA free water (FIG. 1). Specifically, in group 4 of FIG. 1, the cleavage rate on the first day of culture in pig nuclear transfer cloned eggs microinjected with miRNA-148a analogue was 52%, whereas in the control group it was 39.1% and 19.2% when microinjected with miRNA-148a inhibitor. was found to be %. In addition, it is judged that the half-life of miRNA-148a is maintained for about 1 day.

The high cleavage rate on the first day of culture may be because the half-life of miRNA-148a lasts only one day, and this characteristic may affect only the very early stage.

This suggests that the cleavage rate up to the first day of culture can be increased by directly injecting miRNA-148a after fusion of somatic cells and eggs through electrical stimulation when culturing pig nuclear transfer cloned eggs.

Claims (22)

1) removing the nucleus from the mature egg;
2) Obtaining donor cells and injecting them into the perivitelline space of the oocyte from which the nucleus was removed in step 1);
3) fusing the cytoplasm and nucleus of the donor cell by activating the oocyte by electrical stimulation twice;
4) directly injecting microRNA (miRNA)-148a into the cytoplasm of the nuclear-transferred egg obtained by fusion in step 3); and
5) culturing the directly injected cells to increase the initial cleavage rate of nuclear-transferred eggs;
According to claim 1,
A method for improving the development rate of nuclear-transferred eggs using mature eggs obtained in step 1) through in vitro maturation (IVM) for 30 to 70 hours.
According to claim 2,
The method of improving the development rate of nuclear transfer eggs in which the eggs are collected from mammals other than humans.
According to claim 3,
The method of improving the development rate of nuclear transfer eggs in which the mammal is a pig.
According to claim 1,
The direct injection of microRNA (miRNA)-148a in step 4) improves the development rate of nuclear-transferred eggs to increase the initial cleavage rate of nuclear-transferred eggs.
According to claim 5,
The initial method for improving the development rate of nuclear transfer eggs, which is the first day of culture after direct injection of microRNA (miRNA)-148a.

delete delete According to claim 2,
A method for improving the development rate of nuclear transfer eggs in which the mature eggs are treated with hyaluronidase and then pipetted with micromanipulation culture medium to remove cumulus cells.
According to claim 9,
A method for improving the development rate of nuclear-transferred eggs by transferring the oocytes from which the cumulus cells have been removed to a micromanipulated culture medium and selecting them.
The method of claim 9 or 10,
The microengineered culture is a nuclear-transferred egg containing TCM199, 0.001 to 0.01% HEPES, 0.01 to 0.5% BSA, 0.001 to 0.005% penicillin G, and 0.001 to 0.006% streptomycin. How to improve development rate.
According to claim 10,
The method of improving the development rate of nuclear transfer eggs, wherein the oocytes include metaphase II (MII) stage eggs.
According to claim 12,
A method for improving the development rate of nuclear-transferred eggs in which the eggs are stained with Hoechst 33342 and then the nuclei are extracted using a micromanipulation system.
delete delete According to claim 1,
The donor cell in step 2) is a method for improving the development rate of a nuclear transfer egg, which is a somatic cell of a pig.
According to claim 1,
The method for improving the development rate of nuclear transfer eggs in which the donor cells in step 2) are porcine ear-derived fibroblasts.
According to claim 1,
The electrical stimulation in step 3) is performed under conditions of a voltage of 100 to 200 V, a length of 0.01 to 0.1 ms, and a pulse width of 30 to 70 μs.
According to claim 1,
The electrical stimulation in step 3) is performed under conditions of a voltage of 130 to 160 V, a length of 0.02 to 0.04 ms, and a pulse width of 40 to 60 μs.
According to claim 1,
A method for improving the development rate of nuclear-transferred eggs in which the first and second conditions of electrical stimulation in step 3) are made within the same range.
delete delete

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