CN109234225B - Embryo culture solution and method for processing somatic cell cloned embryos - Google Patents

Abstract

The invention discloses an embryo culture solution which contains 200-400 nM of a compound shown as a structural formula (1). The embryo culture solution has obvious effect, and the blastocyst rate of cloned embryos is remarkably improved, so that the efficiency of a cloning technology is improved. The invention also discloses a processing method of the somatic cell cloned embryo, which has simple operation and obvious effect and can remarkably improve the in vitro development efficiency of somatic cell nuclear transfer embryos of cloned embryos of pigs, cows, sheep, mice and humans.

Description

Embryo culture solution and method for processing somatic cell cloned embryos

Technical Field

The invention relates to the technical field of cell in-vitro cloning, in particular to an embryo culture solution and a processing method of somatic cell cloned embryos.

Background

Although many animals have been obtained from adult individuals by somatic cloning techniques, the inefficiency of the technique remains a major cause of limited application. Current research shows that the low efficiency is mainly caused by the reprogramming abnormality of cloned embryos, such as low histone acetylation and high DNA methylation. Most reprogramming of cloned embryonic donor nuclei occurs in early embryos before embryonic genome activation, and histone acetylation is an epigenetic modification and one of the key factors for embryonic gene activation. Histone acetyltransferases act on histones to acetylate them, resulting in a loose chromatin conformation, allowing access to transcription factors, thereby allowing transcription to occur, activating gene reprogramming. The action of histone deacetylase is opposite, and histone deacetylation can be realized, transcription is inhibited, and embryo reprogramming efficiency is reduced. The cloned embryo is constructed by using the fully differentiated somatic cell, the somatic cell is directionally transcribed, the histone deacetylase content of the somatic cell is higher, and the transcriptional activation and the gene expression are inhibited, so that the cloning efficiency is low, and the inhibition of the histone deacetylase is an effective means for improving the cloning efficiency.

Disclosure of Invention

An object of the present invention is to provide an embryo culture solution. Another objective of the invention is to provide a method for processing somatic cloned embryos.

According to one aspect of the present invention, there is provided an embryo culture solution comprising 200 to 400nM of a compound represented by the structural formula (1):

the embryo culture solution has obvious effect, and the blastocyst rate of cloned embryos is remarkably improved, so that the efficiency of a cloning technology is improved.

In some embodiments, the embryo culture fluid contains 200nM, 300nM, or 400nM of the above-described compounds.

Preferably, the embryo culture solution comprises the following components in parts by weight:

wherein LAQ824 is the above compound.

According to one aspect of the present invention, there is provided a method for processing somatic cloned embryos, comprising the steps of:

s1, activating a nuclear transfer reconstructed embryo;

s2, transferring the embryos into the embryo culture solution for culturing for 24 hours;

s3, replacing the culture solution with a common embryo culture solution, and continuing to culture for 168 h.

Cloned embryos may include cloned embryos from all mammals, including but not limited to, pigs, cattle, sheep, mice, and humans.

In some embodiments, the step of S1 includes: removing polar body and nearby cytoplasm by using a micromanipulator from the oocyte, staining for 2min by using fluorescent dye, observing under a fluorescent microscope, discarding the oocyte without completely removing nucleus, injecting a donor cell into the perivitelline space of the completely removed oocyte to complete reconstructed embryo, fusing the reconstructed embryo by using pulses of 85v/mm, 60 mu s and 2 times after the reconstructed embryo is placed in an electric fusion liquid, transferring the reconstructed embryo into an embryo culture liquid to incubate for 1h, and selecting the fused reconstructed embryo to activate by using pulse direct current of 80v/mm, 80 mu s and 2 times.

In some embodiments, the oocyte is collected by:

collecting ovaries from slaughterhouse sow, transferring into 37 deg.C normal saline, washing with normal saline containing antibiotics for 3 times, extracting 2-6mm follicle with 10mL syringe equipped with 18G needle, picking up cumulus oophorus-oocyte complex with self-made ovum pickup needle under stereomicroscope, washing with ovum washing solution for 3 times, washing with mature culture solution for 2 times, and placing into CO₂In mature culture solution with balance of more than 4h in incubator, at 39 deg.C and 5% CO₂And carrying out mature culture for 44h in an incubator with saturated humidity; mixing the mature cumulus-oocyte complex with 0.1% hyaluronidase, repeatedly blowing and beating with a pipette to remove cumulus cells, and selecting oocytes with obvious perivitelline space, no impurities, uniform cytoplasm and obviously discharged first polar body from the oocytes with the cumulus cells removed for construction of cloned embryos.

In some embodiments, the donor cells are harvested by:

cutting pig ear skin, placing into DMEM culture solution at 4 deg.C, storing, transporting to laboratory, cutting pig ear skin tissue pieces, cleaning tissue pieces with DMEM, re-suspending with appropriate amount of fetal calf serum, transferring into culture dish, and culturing at 37 deg.C and 5% CO₂And after culturing for 5-7h in a saturated humidity environment, adding DMEM culture solution containing 10% fetal calf serum, carrying out passage when the cells grow to 90% confluence, and using the somatic cells transferred to the 2 nd generation as donor cells for nuclear transplantation.

The method for processing the somatic cell cloned embryos is simple to operate and obvious in effect, and can remarkably improve the in vitro development efficiency of somatic cell nuclear transfer embryos of cloned embryos of pigs, cows, sheep, mice and humans.

Drawings

FIG. 1 is a micrograph of blastocyst development and blastocyst Hoechst33342 staining after culturing for 24 hours in a culture medium of LAQ824 of 200nM and a control group.

Detailed Description

The present invention will be described in further detail below with reference to the accompanying drawings by taking the construction of a porcine nuclear transfer embryo as an example.

1. Donor cell isolation and culture

The donor cells are derived from ear tissues of Guangdong Wen's group special-grade boars, the pig ear skin is cut and put into DMEM culture solution for preservation at 4 ℃ and then transported back to a laboratory, the pig ear skin tissue blocks are cut into pieces, the DMEM is used for cleaning tissue fragments, then an appropriate amount of fetal calf serum is used for resuspension and is transferred into a culture dish, the temperature is 37 ℃, and the CO content is 5 percent₂And culturing in a saturated humidity environment. After 5-7h, DMEM culture solution containing 10% fetal calf serum is added, and when the cells grow to 90% confluence, the cells are passaged, and the somatic cells transferred to the 2 nd generation are used as donor cells for nuclear transplantation.

2. Oocyte collection and maturation culture

Collecting ovaries from slaughter sows, transferring into 37 deg.C normal saline, washing with normal saline containing antibiotics for 3 times, extracting 2-6mm follicle with 10mL syringe equipped with 18G needle, and picking with home-made ovum picking needle under stereomicroscopeCollecting cumulus-oocyte complexes (COCs), washing with ovum-washing solution for 3 times, washing with mature culture solution for 2 times, and adding CO₂In mature culture solution with balance of more than 4h in incubator, at 39 deg.C and 5% CO₂And carrying out maturation culture for 44h in an incubator with saturated humidity. And mixing the mature cultured COCs with 0.1% hyaluronidase, repeatedly blowing and beating by using a pipette to remove cumulus cells, and selecting oocytes which have obvious perivitelline gaps, no impurities, uniform cytoplasm and obviously discharged first polar bodies from the oocytes without the cumulus cells to construct cloned embryos.

3. Somatic cell nuclear transfer

And removing the polar body and about 15% of cytoplasm nearby the mature oocyte by using a micromanipulator, then staining for 2min by using a fluorescent dye, observing under a fluorescent microscope, discarding the oocyte without completely removing the nucleus, and completely injecting a donor cell into the perivitelline space to complete the embryo reconstruction process. After the reconstructed embryo is put into the electric fusion liquid, the reconstructed embryo is fused by 2 pulses of 85v/mm and 60 mu s, then the reconstructed embryo is transferred into the embryo culture liquid to be incubated for 1h, the fused embryo is selected to be activated by 2 pulse direct currents of 80v/mm and 80 mu s, and the activated embryo is cultured in groups.

Control group: the somatic cell cloned embryos are put into an embryo culture solution to be cultured for 168 hours.

Experimental groups: the cloned embryos were cultured in medium containing LAQ824 at 100nM, 200nM, 300nM and 400nM for 24h, then transferred to embryo culture medium and cultured continuously for 168 h.

In this example, the embryo culture solution comprises the following components in parts by weight:

wherein, the chemical formula of LAQ824 is: c₂₂H₂₅N₃O₃。

LAQ824 has the structural formula:

4. in vitro culture and blastocyst cell counting of cloned embryos

The culture conditions of cloned embryo are 39 ℃ and 5% O₂、5％CO₂、90％N₂And saturation humidity, observing and recording the development conditions of the cleavage and the blastocyst on the 2 nd day and the 7 th day of culture respectively, fixing the blastocyst on the 7 th day by using 4% paraformaldehyde for 10min, then dyeing in 10mg/L Hoechst33342 for 10min, and observing and recording the number of blastocyst cells under a fluorescence microscope after tabletting. A micrograph of blastocyst development and blastocyst Hoechst33342 staining, in which the control group was cultured in 200nM of LAQ824 for 24h, is shown in FIG. 1.

Wherein, A: a blastocyst development micrograph of a control group;

b: blastocyst development micrographs cultured in 200nM LAQ824 for 24 h;

c, a microscopic staining picture of a blastocyst of a control group, Hoechst 33342;

a24 h blastocyst was cultured in a culture medium of LAQ824 from D200nM in Hoechst33342 staining photomicrograph.

Analysis of variance was performed on the experimental data using SPSS software, comparing the early development of the treated, treated and untreated embryos at different concentrations, including the differential results of embryo cleavage rate, blastocyst rate and total blastocyst cell count, as reported in tables 1 and 2.

TABLE 1 Effect of different concentrations of LAQ824 on the efficiency of in vitro development of cloned embryos

Remarking: data from 3 replicates were statistically analyzed and the mean. + -. standard error was calculated, with different lower case letters in the same column indicating significant differences (P < 0.05), as follows.

TABLE 2 influence of LAQ824 treatment of cloned embryos at different times on their in vitro developmental efficiency

The result shows that the in vitro development efficiency of the porcine cloned embryo can be obviously improved by treating the porcine somatic cloned embryo for 24h by using 200nM LAQ824, and the blastocyst rate of 168h is extremely higher than that of a control group.

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.

Claims (5)

1. An embryo culture solution is characterized by comprising the following components in percentage by weight:

NaCl 108 mM；

KCl 10 mM；

KH₂PO₄ 0.35 mM；

MgSO₄·7H₂O 0.40 mM；

NaHCO₃ 25.07 mM；

Na-pyruvate 0.20 mM；

Ca-(lactate)2·5H₂O 2.00 mM；

L-Glutamine 2.00 mM；

Hypotaurine 5.00 mM；

EAA 20.00 mL/L；

NEAA 10.00 mL/L；

BSA 4.00 mg/mL；

LAQ824 200 nM；

wherein, the LAQ824 is a compound shown as a structural formula (1):

（1）。

2. a method for processing porcine somatic cell cloned embryos is characterized by comprising the following steps:

s1, activating a nuclear transfer reconstructed embryo;

s2, transferring the embryos into the embryo culture solution of claim 1 and culturing for 24 hours;

s3, replacing the culture solution with the culture solution containing other components except the LAQ824 as the claim 1, and continuing culturing for 168 h.

3. The method for processing somatic cloned embryos of claim 2, wherein the step of S1 comprises: removing polar body and nearby cytoplasm by using a micromanipulator, staining the oocyte for 2min by using a fluorescent dye, observing under a fluorescent microscope, discarding the oocyte without completely removing nucleus, injecting a donor cell into the perivitelline space of the oocyte with completely removed nucleus to complete a reconstructed embryo, fusing the reconstructed embryo by using 85v/mm, 60 mu s and 2 pulses after the reconstructed embryo is placed into an electric fusion liquid, transferring the reconstructed embryo into a culture solution containing other components except LAQ824 components and described in claim 1 for incubation for 1h, and selecting the fused reconstructed embryo to be activated by using 80v/mm, 80 mu s and 2 pulses of direct current.

4. The method for processing somatic cloned embryos of claim 3, wherein the oocytes are collected by:

collecting ovaries from slaughterhouse sow, transferring into 37 deg.C normal saline, washing with normal saline containing antibiotics for 3 times, extracting 2-6mm follicle with 10mL syringe equipped with 18G needle, picking up cumulus oophorus-oocyte complex with self-made ovum pickup needle under stereomicroscope, washing with ovum washing solution for 3 times, washing with mature culture solution for 2 times, and placing into CO₂In mature culture solution with balance of more than 4h in incubator, at 39 deg.C and 5% CO₂And carrying out mature culture for 44h in an incubator with saturated humidity; mixing the mature cumulus-oocyte complex with 0.1% hyaluronidase, repeatedly blowing and beating with a pipette to remove cumulus cells, and selecting oocytes with obvious perivitelline space, no impurities, uniform cytoplasm and obviously discharged first polar body from the oocytes with the cumulus cells removed for construction of cloned embryos.

5. The method of claim 3, wherein the donor cells are harvested by:

cutting pig ear skin, placing into DMEM culture solution at 4 deg.C, storing, transporting to laboratory, cutting pig ear skin tissue pieces, cleaning tissue pieces with DMEM, re-suspending with appropriate amount of fetal calf serum, transferring into culture dish, and culturing at 37 deg.C and 5% CO₂And after culturing for 5-7h in a saturated humidity environment, adding DMEM culture solution containing 10% fetal calf serum, carrying out passage when the cells grow to 90% confluence, and using the somatic cells transferred to the 2 nd generation as donor cells for nuclear transplantation.

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