CN107299113A - Application processes of the H3K27me3 and its demethylase KDM6A/B in mouse nuclear transfer reconstructed embryo - Google Patents

Abstract

The invention discloses an application method of H3K27me3 and its demethylase KDM6A/B in mouse nuclear transfer reconstituted embryos, and analyzes the H3K27 trimethylation pattern and cloned embryos in parthenogenetic embryos at various stages before implantation What is the difference, the impact of this difference in the pattern on the developmental ability of cloned embryos, confirmed that the fine-tuning of KDM6A and KDM6B can improve the blastocyst development rate of cloned embryos and the birth rate of cloned animals.

Description

Expression of H3K27me3 and its demethylase KDM6A/B in embryos reconstituted by nuclear transfer in mice application method

technical field

The invention relates to the field of biotechnology, in particular to an application method of H3K27me3 and its demethylase KDM6A/B in mouse nuclear transfer reconstituted embryos.

Background technique

Somatic cell nuclear transfer refers to the technique of injecting somatic cell nuclei into the cytoplasm of enucleated oocytes to obtain reconstructed embryos, which eventually develop into offspring; cell reprogramming can be achieved through nuclear transfer, cell fusion, specific transcription factors, and culture However, at present, the only reprogramming method that can induce cells to redevelop into individuals is somatic cell nuclear transfer. Other methods can only produce induction at the cellular, molecular or biochemical levels, and all have some shortcomings. Since the establishment of somatic cell nuclear transfer technology, the cloning efficiency has been at a low level, only a few reconstituted embryos can develop to full-term birth, usually only 1-2% birth rate in mice, and most cloned mice It will die shortly after birth, and the successfully born cloned mice will also have abnormal symptoms such as placenta enlargement and fetal overgrowth, which is the so-called "fetal macrosomia". The main reason for this phenomenon is that the donor cell nucleus cannot be absorbed It is caused by complete reprogramming; reprogramming mainly refers to the erasure and reconstruction of epigenetic modification. An epigenetic modification pattern that is forcibly reversed to the embryonic state by the oocyte cytoplasm in a very short period of time before genome activation. When the donor nucleus is injected into the enucleated oocyte, the reprogramming process begins, which mainly includes rearrangement of nuclear and extranuclear structures, DNA methylation, genome imprinting, X chromosome activity, histone methylation/ "Erasure and rebuild" of a series of epigenetic modifications such as acetyl/phosphorylation and telomere length.

Since the success of somatic cell reprogramming mediated by nuclear transfer technology, the research on nuclear transfer technology has always revolved around the core issue of how to improve the reprogramming efficiency. Nuclear transfer mediated reprogramming is usually evaluated from the following aspects : (1) blastocyst development rate of reconstituted embryos; (2) birth rate of cloned animals; (3) survival rate of cloned animals; (4) success rate of obtaining embryonic stem cells from reconstituted blastocysts; in 2006, Japanese scientists Takahashi and Yamanaka used IPS technology to arrange and combine 24 candidate genes and screen them one by one, and finally determined the key factors that can induce body weight programming, Oct-3/4, Sox2, c-Myc and Klf4. Factors capable of restoring mouse fibroblasts to a pluripotent state.

Somatic cell nuclear transfer is the only technical method that can restore terminally differentiated somatic cells to the state of embryonic cells and develop into individuals. After the somatic cell nucleus enters the oocyte cytoplasm, it can reprogram the donor nucleus in a very short time and return to the embryonic state. In the state of totipotency, the cytoplasmic reprogramming ability of oocytes to somatic cells is much higher than that of specific transcription factors transferred from exogenous sources. In nuclear transfer reconstituted embryos, the expression of originally stable and terminally differentiated somatic cells The epigenetic modification mode is forcibly reversed by the oocyte cytoplasm to the epigenetic modification mode of the embryonic state in a very short period of time before the zygotic genome is activated. When the donor nucleus is injected into the enucleated oocyte, the reprogramming process begins. This process mainly includes nuclear and extranuclear structural rearrangement, DNA methylation, genome imprinting, X chromosome activity, histone methyl/acetyl/phosphorylation, and telomere length and a series of epigenetic modification "wiping" Removal and Reconstruction".

During the early development of mouse embryos, the epigenetic modification is also undergoing drastic changes. This dynamic change is in preparation for the correct activation and silencing of specific genes. The type and degree of epigenetic modification on histones , directly determines whether the zygote genome can be successfully activated; histone methylation modification is an important epigenetic modification, histone methylation modification is related to the formation of heterosomes, the inactivation of X chromosome, and the transcription of specific genes Regulation, genome integrity, and cell development are closely related, and this modification plays a very important role in early embryonic development; current studies have shown that H3K9me3, H3K27me3, and H3K72me3 are signs of DNA transcriptional repression; while H3K4me3 , H3K36me3 and H3K79me3 are related to the transcriptional activation of genes. H3K27me3 modification can remove methyl groups through two histone demethylases KDM6A and KDM6B. Both KDM6A and KDM6B have Jmjc dioxygenase domains, which are The active center of demethylation, KDM6A, KDM6B, the expression patterns of two important histone demethylases in mouse embryos; what is the relationship between KDM6A, KDM6B and H3K27me3 modification in oocytes; and The relevant research has not been reported yet. In order to solve the above problems, embryos at various stages of parthenogenety activation were used as the research object to study the relationship between H3K27me3, KDM6A and KDM6B in terms of time and function, so as to find ways to improve the developmental ability of embryos. is the current research direction.

With the research on the molecular mechanism of pluripotency of embryonic stem cells, the in-depth understanding of the mechanism of reprogramming has been promoted; the study found that Oct4, Sox2, and Nanog combine with each other to form a complex, establishing a self-regulatory feedback mechanism; reprogramming factors and The relationship between H3K27me3 is shown in Figure 1; it can be seen from the figure that the target genes of reprogramming transcription factors are mainly divided into three categories: (1) Genes actively expressed in embryonic stem cells, during the reprogramming process, most of the genes that inhibit expression Genes colocalize with PcG, and the PcG protein family is an important part of the PRC complex, the most important of which is PRC2 catalyzing the trimethylation of lysine 27 of histone H3, and the expression of genes with H3K27me3 modification will be inhibited ; (2) The genes that are repressed in embryonic stem cells, in reprogramming, the trimethylation modification of the 4th lysine of histone H3 in the promoter region of certain stemness genes, transcription factors can be re-recruited to The promoter regions of these genes activate the expression of stemness genes; (3) the key genes in the "equilibrium state" will form a unique chromosomal modification of pluripotent stem cells during reprogramming, which is also transcriptionally active H3K4me3 and repressed H3K27me3 mark, the "bivalent domain", this unique epigenetic modification maintains the plasticity of key genes, allowing pluripotent cells to have multiple choices during differentiation.

Histone methylation is not only closely related to heterochromosomal formation, X chromosome inactivation, gene transcription regulation, and genome integrity, but also plays an important role in somatic cell reprogramming and early embryonic development. Among them, H3K27me3 This modification can remove the methyl group by two demethylases: (1) KDM6A, also known as UTX; (2) KDM6B, also known as JMJD3, KDM6A and KDM6B remove the H3K27me3 modification of specific genes during reprogramming , so that the gene is activated. The KDM6A gene is located on the X chromosome, contains six TPRs, and can escape the inactivation of the X chromosome. Both KDM6A and KDM6B contain a very conserved JmjC domain. and α-ketoglutarate to complete the demethylation process.

It has been reported that in mouse embryonic development and cell differentiation, KDM6A has an important regulatory effect on key genes affecting embryonic development; like KDM6A, KDM6B is also an important demethylase of H3K27me3, encoded by autosomes, in Studies on mice have shown that KDM6B is involved in the transformation of white and brown fat by regulating the activity of key fat genes; after bone injury, KDM6B can regulate the differentiation of osteoclasts and may limit the pathogenesis of osteoporosis, and is associated with There is a certain relationship between skin repair; when nerve cells are damaged, KDM6B can limit the excessive proliferation and regeneration of Schwann cells, thereby preventing the generation of neurofibromas. In addition, KDM6B also plays a regulatory role in pro-inflammatory and anti-inflammatory responses. In summary, the demethylase activity of KDM6A and KDM6B is very important in cell differentiation, not only involved in cell cycle regulation, macrophage differentiation, neural stem cell differentiation, but also can regulate cell reprogramming process, participate in embryonic stem cell differentiation , regulating the expression of genes related to embryonic development and other aspects, H3K27me3 histone demethylase KDM6A/B was discovered in 2007, and related research mainly focused on the biochemical mechanism of demethylation, studying KDM6A/B Its role in the development of nuclear transfer reconstituted embryos has not been reported yet.

Contents of the invention

The purpose of the present invention is to provide an application method of H3K27me3 and its demethylase KDM6A/B in mouse nuclear transfer reconstituted embryos, so as to solve the problems raised in the above-mentioned background technology.

To achieve the above object, the present invention provides the following technical solutions:

A method for applying H3K27me3 and its demethylase KDM6A/B in mouse nuclear transfer reconstituted embryos, comprising the following steps:

1) Prepare experimental animals:

Use Oct4 promoter-green fluorescent transgenic mice as oocyte donor mice (OG2) for live cell imaging;

2) Collection of MII stage oocytes:

3) Prepare experimental reagents:

3.1) Embryo culture medium:

3.2) Main reagents related to immunofluorescence and western blotting:

4% paraformaldehyde solution, polyethylene glycol octylphenyl ether, phosphate-buffered saline, Tween 20, DAPI dye, antibody Oct4, antibody Nanog, antibody SSEA1, antibody Sox2, antibody H3K27me3, antibody KDM6A, antibody KDM6B , RNA extraction kit, real-time fluorescent quantitative PCR;

4) Quantitative primers

4.1) KDM6A, the primer sequence is:

forward-TATTGGCCCAGGTGACTGTGAA

reverse-CAGATCTCCAGGTCGCTGAATAAAC

4.2) KDM6B, the primer sequence is:

forward-GCTGGAGTGCTTGTTCCATGAG

reverse-GAAAGCCAATCATCACCCTTGTC

4.3) Gapdh, the primer sequence is:

forward-AAAATGGTGAAGGTCGGTGTG

reverse-AATGAAGGGGTCGTTGATGG

5) Experimental method

5.1) Parthenogenetic activation of MII stage oocytes

5.2) siRNA microinjection

Dilute siRNA-KDM6A, siRNA-KDM6B, and siRNA-control group to 40mM with nuclease-free TE buffer, and slowly inject the diluted liquid into the oocyte cytoplasm through a microinjection needle using a micromanipulator After the injection, the oocytes were recovered at room temperature for 15 minutes, then transferred to KSOM medium, and placed in a 37°C incubator for 2 hours;

5.3) Real-time quantitative PCR, according to the recommendations of the quantitative instructions, conduct experiments;

5.4) Immunofluorescence staining

5.4.1) Embryo fixation: put the embryos into a 4% paraformaldehyde fixative solution, and fix them overnight at room temperature for 60 minutes or at 4°C;

5.4.2) Permeabilization: Wash the fixed embryos with 1ml of phosphate-buffered saline, transfer to 0.5% polyethylene glycol octylphenyl ether solution and treat at room temperature for 15 minutes;

5.4.3) Sealing: put the embryo into a solution containing 0.2% polyethylene glycol octyl phenyl ether and 1% bovine serum albumin to seal at room temperature for 30 minutes;

5.4.4) Antibody incubation: transfer the embryos into the antibody diluted with the diluent and incubate at 37°C for 1 h. The diluent contains 1% Tween 20 and 1% bovine serum albumin. After incubation, use Rinse with phosphate buffered saline for 3 times, 5 min each time, add antibody solution diluted in blocking solution, containing 0.2% polyethylene glycol octylphenyl ether and 1% bovine serum albumin in the blocking solution Solution, incubate at room temperature for 1 hour in the dark, and then rinse with phosphate-buffered saline in the dark for 3 times, 5 minutes each time;

5.4.5) DAPI staining: stain the embryos at room temperature in the dark for 15 minutes, rinse with phosphate-buffered saline in the dark for 3 times, 5 minutes each time;

5.4.6) Sealing: Add a drop of anti-quenching agent on the glass slide, transfer the stained embryos into the anti-quenching agent, cover the coverslip and seal with the mounting agent;

5.4.7) Confocal imaging: use a Nikon A1R confocal microscope to collect images;

5.5) Live cell imaging: oocytes after microinjection of siRNA were parthenogenetically activated for 6 hours, cultured in NikonTi-E with a live cell imaging system, scanned with a 488nm laser, and scanned by a Nikon A1R camera;

5.6) Data statistics and analysis.

As a further solution of the present invention: in the step 1, the mice are purchased from the Experimental Animal Research Center of Inner Mongolia University, and the donor mice are kept in an SPF grade animal room at 22-24°C, with a relative humidity of 50-60%, and the animal room is illuminated. Hours are from 8 am to 8 pm daily.

As a further solution of the present invention: in the step 3.1, the preparation method of the embryo culture solution is:

1) HCZB concentrated stock solution: 500mL; composition: 495ml ultrapure water, 2380mg sodium chloride, 180mg potassium chloride, 145mg magnesium sulfate heptahydrate, 20mg disodium edetate, 2.65mL sodium lactate, 500mg glucose, Potassium dihydrogen phosphate 80mg;

2) 10ml calcium chloride 100X concentrated stock solution: prepared from 0.2g calcium chloride dihydrate and 10ml ultrapure water;

3) 10ml strontium chloride 100X concentrated stock solution: prepared from 2.666g strontium chloride hexahydrate and 10ml ultrapure water;

4) 20ml glutamine 100X concentrated stock solution: prepared from 0.294g glutamine and 20ml calcium-free CZB solution;

5) Cytochalasin B solution: prepared by dissolving cytochalasin B in a dimethyl sulfoxide solution at a ratio of 1 mg/ml;

6) Polyvinylpyrrolidone solution:

PVP solution with a mass fraction of 10%: prepared from 1g PVP and 10ml HCZB;

PVP solution with a mass fraction of 3%: prepared from 0.3g PVP and 10ml HCZB;

7) Hyaluronidase solution: prepared from 20ml of M2 culture medium and 100mg of hyaluronidase.

As a further solution of the present invention: in the step 5.2, the preparation method of the KSOM culture solution is as follows: every 100mL KSOM-AA embryo culture solution contains 0.38mg of disodium salt of edetate, 559.5mg of sodium chloride, Potassium 18.5mg, potassium dihydrogen phosphate 4.75mg, magnesium sulfate heptahydrate 4.95mg, sodium lactate solution 0.174ml, glucose 3.60mg, sodium bicarbonate 210.0mg, glutamine 14.5mg, sodium pyruvate 2.2mg, double antibody 1 6.3 mg, double antibody 2 5.0mg, essential amino acid 1.0ml, non-essential amino acid 0.5ml; ethylenediaminetetraacetic acid disodium salt was added dropwise first, 100mg bovine serum albumin was added to 100mL KSOM-AA embryo culture medium, gently Mix well to slowly dissolve the bovine serum albumin, do not shake the liquid violently to avoid foaming and protein denaturation; use a 0.22μm needle filter to filter the culture solution into a sterile plastic tube, store the solution at 4°C, and store for 1-2 2 weeks.

As a further solution of the present invention: in the step 5.6, the data statistics and analysis method adopts SPSS10.0 statistical software analysis and processing, P<0.05 means significant difference, and P<0.01 means extremely significant difference.

Compared with the prior art, the beneficial effect of the present invention is: the present invention detects the modification of H3K27me3 in mouse parthenogenetic embryos at different stages, and the results show that in mouse 8-cell and morula, no Modification of H3K27me3; in pre-implantation embryos, KDM6A and KDM6B are highly expressed, but KDM6A protein cannot be detected in 8-cell stage embryos; in pre-implantation embryos, KDM6A and KDM6B exist at the mRNA and protein levels The phenomenon of functional compensation; reducing the expression of KDM6B helps to improve the blastocyst rate and blastocyst quality of embryos, that is, 95.1% vs. 84.3%, P<0.05, the reduction of KDM6B expression helps Oct4-GFP The expression of , thus contributing to the improvement of oocyte reprogramming ability; the present invention uses mice as the research object, and analyzes the differences between the H3K27 trimethylation patterns in parthenogenetic embryos at various stages before implantation and cloned embryos, as well as the differences The impact on the developmental ability of cloned embryos, the results show that increasing the expression of KDM6A and/or reducing the expression of KDM6B can help to improve the blastocyst rate, blastocyst and embryonic stem cell quality of nuclear transfer; fine-tuning of KDM6A and KDM6B can improve The blastocyst development rate of cloned embryos and the birth rate of cloned animals pave the way for the further development of somatic cell nuclear transfer technology.

Description of drawings

Figure 1 is a schematic diagram of the dynamic changes of H3K27me3 in mouse pre-implantation embryos - each developmental stage of mouse parthenogenetic activation embryos, and the scale bar is 10 μm.

Figure 2 is a schematic diagram of the dynamic changes of H3K27me3 in mouse pre-implantation embryos - H3K27me3 immunofluorescence staining results at 2-cell, 4-cell, 8-cell, morula and blastocyst stages, the scale bar is 10 μm.

Figure 3 shows the dynamic changes of H3K27me3 in mouse pre-implantation embryos - the H3K27me3 fluorescence signal only exists in the inner cell mass in hatched blastocysts, and the scale bar is 10 μm.

Fig. 4 is a columnar comparison chart of expression patterns of KDM6A and KDM6B genes in MII oocytes before enucleation and after enucleation.

Fig. 5 is a graph showing the changes of KDM6A at various stages before implantation detected by real-time quantitative PCR.

Fig. 6 is a graph showing the changes of KDM6B at various stages before implantation detected by real-time quantitative PCR.

Figure 7 is a map of the cellular localization of KDM6A and KDM6B in MII stage oocytes. The scale bar is 50 μm in the upper panel and 20 μm in the lower panel.

Figure 8 is a map of the localization of KDM6A in 2-cell, 4-cell, 8-cell, morula and blastocyst analyzed by immunofluorescence staining, and the length of the scale bar is 20 μm.

Figure 9 is a map of the localization of KDM6B in 2-cell, 4-cell, 8-cell, morula and blastocyst analyzed by immunofluorescence staining, and the length of the scale bar is 20 μm.

Fig. 10 is a comparison chart of KDM6A and KDM6B proteins detected in MII stage oocytes by immunoblotting.

Figure 11 is a map of the subcellular localization of KDM6A in fetal fibroblasts, the length of the scale bar is 50 μm.

Figure 12 is a map of the subcellular localization of KDM6B in fetal fibroblasts, the length of the scale bar is 50 μm.

Fig. 13 is a schematic diagram of the location of KDM6A-siRNA interference.

Fig. 14 is a schematic diagram of the location of KDM6B-siRNA interference.

Figure 15 is a schematic diagram of the microinjection of siRNA into MII stage oocytes and the time points of embryo collection.

Fig. 16 is a bar graph of the determination results of the real-time quantitative PCR to verify the specificity of the siRNA interference fragment and the efficiency of the interference efficiency.

Figure 17 is a graph showing the changes of H3K27me3 in 8-cell, morula stage embryos after single and double interference KDM6A and KDM6B were detected by immunofluorescence.

Figure 18 is a graph showing the changes of H3K27me3 in morula stage embryos after single and double interference KDM6A and KDM6B detected by immunofluorescence.

Fig. 19 is a graph showing the expression of KDM6B mRNA after KDM6A-siRNA injection by real-time quantitative PCR.

Fig. 20 is a graph showing the expression of KDM6A mRNA after KDM6B-siRNA injection by real-time quantitative PCR.

Fig. 21 is a diagram showing the protein changes of KDM6A and KDM6B detected by immunoblotting after siRNA injection.

Fig. 22 is a bar graph of the protein changes of KDM6A and KDM6B after siRNA injection detected by immunoblotting.

Figure 23 shows the effects of interfering with KDM6A and KDM6B on blastocyst quality, the scale bar is 100 μm.

Fig. 24 is a statistical histogram of the blastocyst development rate of embryos injected with siRNA.

Fig. 25 is an electron microscope image of blastocyst nuclei stained with DAPI after siRNA injection, and the scale bar in the figure is 50 μm.

Figure 26 is a schematic diagram of the statistical results of the number of blastocyst cells stained with DAPI nuclei, **P<0.01, ***P<0.001.

Figure 27 is a schematic diagram of the detection results of OG2 transgenic mice detected by nested PCR. The mice with a single band at 200 bp are Oct4-GFP homozygotes.

Figure 28 is a 488nm laser scan to detect the GFP fluorescence signal in the blastocyst after interference with KDM6B, the scale bar before interference is 50 μm, and the scale bar after interference is 20 μm.

Fig. 29 is a time-lapse photographed image of embryos injected with Control-siRNA through live cells, taken under magnification of 200X.

Fig. 30 is a live cell time-lapse photographed image of an embryo injected with KDM6B-siRNA, taken at a magnification of 200X.

detailed description

The technical solution of this patent will be further described in detail below in conjunction with specific embodiments.

Please refer to Figure 1-30, a method for applying H3K27me3 and its demethylase KDM6A/B in mouse nuclear transfer reconstituted embryos, including the following steps:

1) Prepare experimental animals:

Use Oct4 promoter-green fluorescent transgenic mice as oocyte donor mice (OG2) for live cell imaging;

2) Collection of MII stage oocytes:

3) Prepare experimental reagents:

3.1) Embryo culture medium:

3.2) Main reagents related to immunofluorescence and western blotting:

4% paraformaldehyde solution (PFA), polyethylene glycol octylphenyl ether, phosphate buffered saline (PBS), Tween 20, DAPI dye, antibody (Oct4) (dilution ratio 1:500), antibody ( Nanog) (dilution ratio 1:500), antibody (SSEA1) (dilution ratio 1:50), antibody (Sox2) (dilution ratio 1:500), antibody (H3K27me3) (dilution ratio 1:50), antibody (KDM6A) (dilution ratio 1:50), antibody (KDM6B) (dilution ratio 1:500), RNA extraction kit, real-time fluorescent quantitative PCR;

4) Quantitative primers

4.1) KDM6A, the primer sequence is:

forward-TATTGGCCCAGGTGACTGTGAA

reverse-CAGATCTCCAGGTCGCTGAATAAAC

4.2) KDM6B, the primer sequence is:

forward-GCTGGAGTGCTTGTTCCATGAG

reverse-GAAAGCCAATCATCACCCTTGTC

4.3) Gapdh, the primer sequence is:

forward-AAAATGGTGAAGGTCGGTGTG

reverse-AATGAAGGGGTCGTTGATGG

5) Experimental method

5.1) Parthenogenetic activation of MII stage oocytes

The collection time point of parthenogenetically activated embryos is shown in Figure 1.

5.2) siRNA microinjection

The siRNA-KDM6A, siRNA-KDM6B and siRNA-control group were diluted to 40mM with nuclease-free TE buffer, and the diluted liquid was slowly injected into the oocyte cytoplasm through a microinjection needle using a micromanipulator After the injection, the oocytes were recovered at room temperature for 15 minutes, then transferred to KSOM medium, and placed in a 37°C incubator for 2 hours;

5.3) Real-time quantitative PCR, according to the recommendations of the quantitative instructions, conduct experiments;

5.4) Immunofluorescence staining

5.4.1) Embryo fixation: put the embryos into a 4% paraformaldehyde fixative solution, and fix them overnight at room temperature for 60 minutes or at 4°C;

5.4.2) Permeabilization: Wash the fixed embryos with 1ml of phosphate-buffered saline, transfer to 0.5% polyethylene glycol octylphenyl ether solution and treat at room temperature for 15 minutes;

5.4.3) Sealing: put the embryo into a solution containing 0.2% polyethylene glycol octyl phenyl ether and 1% bovine serum albumin to seal at room temperature for 30 minutes;

5.4.4) Antibody incubation: transfer the embryos into the antibody diluted with diluent and incubate at 37°C for 1 h. The diluent contains 1% Tween 20 and 1% bovine serum albumin. After incubation, use Rinse with phosphate buffered saline for 3 times, 5 min each time, add antibody solution diluted in blocking solution, containing 0.2% polyethylene glycol octylphenyl ether and 1% bovine serum albumin in mass fraction Solution, incubate at room temperature for 1 hour in the dark, then rinse with phosphate buffered saline in the dark for 3 times, 5 minutes each time;

5.4.5) DAPI staining: stain the embryos at room temperature in the dark for 15 minutes, rinse with phosphate-buffered saline in the dark for 3 times, 5 minutes each time;

5.4.6) Sealing: Add a drop of anti-quenching agent on the glass slide, transfer the stained embryos into the anti-quenching agent, cover the coverslip and seal with the mounting agent;

5.4.7) Confocal imaging: use a Nikon A1R confocal microscope to collect images;

5.5) Live cell imaging: oocytes after microinjection of siRNA were parthenogenetically activated for 6 hours, cultured in NikonTi-E with a live cell imaging system, scanned with a 488nm laser, and scanned by a Nikon A1R camera;

5.6) Data statistics and analysis;

5.7) Results: The dynamic changes of H3K27me3 modification in parthenogenetic embryos are as follows:

Detect the dynamic changes of H3K27me3 in the gynonucleus of mouse embryos, perform parthenogenetic activation on oocytes, collect embryos according to the time points in Figure 1, and separate them in MII, 2-cell, 4-cell, 8-cell, morula, and sac H3K27me3 modification was detected at the stage of embryo and hatched blastocyst, and the results are shown in Figure 2; oocytes at the MII stage already had H3K27me3 modification; the H3K27me3 fluorescence signal began to weaken from the 2-cell stage; at the 8-cell and morula stage, there was no H3K27me3 fluorescence signal; at the blastocyst stage, the cells in the whole embryo have H3K27me3 modification; when the blastocyst hatches, the H3K27me3 fluorescence signal only exists in the inner cell mass, as shown in Figure 3; in the polar body, H3K27me3 modification is not only in There are always embryos at all stages, and the intensity of the fluorescent signal is higher than that of the nucleus in the embryo;

5.7.1) Dynamic changes of demethylase KDM6A and KDM6B mRNA in pre-implantation embryos

By fluorescent quantitative PCR (RT-qPCR) technology, the mRNA content changes of KDM6A and KDM6B in oocytes before and after enucleation were detected, as shown in Figure 4; KDM6A and KDM6B mainly exist in oocyte cytoplasm; by RT -QPCR detects the dynamic changes of KDM6A and KDM6B in embryos at various stages before implantation. When the oocyte is activated, KDM6A and KDM6B immediately begin to degrade, and the mRNA of KDM6A is almost undetectable at the blastocyst stage, as shown in Figure 5, while KDM6B It is close to the lowest point at the 8-cell stage, as shown in Figure 6;

5.7.2) Dynamic changes of KDM6A and KDM6B in pre-implantation embryos

The positions of KDM6A and KDM6B in embryos at different stages were determined by immunofluorescence technique. The protein content of KDM6A and KDM6B was very high in MII oocytes, and KDM6A was mainly concentrated around the spindle, and KDM6B was evenly distributed throughout the MII stage oocytes. In cells, as shown in Figure 7; the detection of KDM6A fluorescence signal disappeared at the 8-cell stage, and at the same time, the fluorescence signal of H3K27me3 could not be detected, as shown in Figure 8; and it can be detected at all stages of embryonic development The fluorescent signal of KDM6B is shown in Figure 9; immunoblotting experiments were used to further confirm the results of immunofluorescence staining, and 1000 oocytes at the MII stage were collected, and Western blot was used to detect the protein expression levels of KDM6A, KDM6B and H3K27me3, respectively, and immunoassay The results of fluorescent staining were consistent with the results of immunofluorescence. Washed hybridization bands appeared at the positions of 154kDa (KDM6A), 180kDa (KDM6B) and 55kDa (H3K27me3), respectively, as shown in Figure 10; in order to ensure the specificity of the antibody, at the same time Immunofluorescent staining was performed in fetal fibroblasts as shown in Figures 11-12.

5.7.3) KDM6A and KDM6B have functional compensation revealed by RNA interference technology

Since both KDM6A and KDM6B have the function of demethylase and are highly expressed in oocytes, it is speculated that KDM6A and KDM6B have a functional compensation mechanism in early embryonic development. In order to verify this conjecture, we designed and siRNA was synthesized to interfere with KDM6A and KDM6B; since the gene often has alternative splicing isoforms in oocytes, in order to prevent the existence of isoforms in KDM6A and KDM6B, two splicing isoforms were designed at 585 and 1,518 bp downstream of the KDM6A promoter, respectively. Two siRNAs are mixed according to 1:1 and called KDM6A-siRNA; similarly, two siRNAs are respectively designed at 4,536 and 4,749bp downstream of the KDM6B promoter, and become KDM6B-siRNA after uniform mixing; at the same time, A control-siRNA that does not affect any endogenous gene was also synthesized and used in the control group, as shown in Figure 13-14; the gene sequence of the control-siRNA is as follows:

KDM6A-1

Sense: GGACUUGCAGCACGAAUUATT

Anti: UAAUUCGUGCUGCAAGUCCAG

KDM6A-2

Sense: CGCUGCUACGAAUCUCUAATT

Anti: UUAGAGAUUCGUAGCAGCGAA

KDM6B-1

Sense: CGUCCAAUAUUCCUGUUUATT

Anti: UAAACAGGAAUAUUGGACGCA

KDM6B-2

Sense: CCGUGCAGCUAUACAUGAATT

Anti: UUCAUGUAUAGCUGCACGGTG

siRNA was microinjected into MII stage oocytes, as shown in Figure 15; in order to verify the effectiveness and specificity of siRNA, the expression levels of KDM6A and KDM6B in oocytes after injection of siRNA were detected by real-time quantitative PCR technology, As shown in Figure 16; real-time quantitative PCR results show that the interference efficiency of KDM6A-siRNA and KDM6B-siRNA is more than 90%, and Control-siRNA has almost no effect on the expression of endogenous gene GAPDH; Immunofluorescence detection of H3K27me3 modification in 8-cell and morula stage embryos was performed, as shown in Figure 17-18; the immunofluorescence results fully demonstrated that interference with either KDM6A or KDM6B alone would not affect the modification of H3K27me3 in embryos, Simultaneously interfering with KDM6A and KDMB6 at the 8-cell and morula stage can cause the fluorescent signal of H3K27me3 to appear in the nucleus; then, for embryos injected with KDM6A-siRNA alone, the mRNA expression of KDM6B was detected at different stages of development, using the same method Method: After injecting KDM6B-siRNA, the mRNA expression of KDM6A is detected, as shown in Figure 19-20; the single interference results show that KDM6A and KDM6B have a compensatory effect at the mRNA level, that is, reducing any one of the two genes The transcription of another gene will increase accordingly; since the mRNA expression of some genes is not synchronized with the protein content in the embryonic stage, in order to rule out this possibility, control-siRNA, KDM6A-siRNA were collected and injected The embryos at the 2-cell stage after KDM6B-siRNA were subjected to immunoblotting experiments, as shown in Figure 21-22; the results of Western blot detection of protein were consistent with the results of Real time PCR detection of mRNA, and the above results confirmed that KDM6A and KDM6B were in the Both mRNA and protein levels have functional compensatory effects.

5.7.4) Effects of KDM6A and KDM6B on embryonic development

In order to study the role of KDM6A and KDM6B in pre-implantation embryos, the embryo development after injection of KDM6A-siRNA and KDM6B-siRNA were counted respectively. The statistical results showed that the cleavage rates of KDM6A and KDM6B embryos were 97.7% and 98.6% respectively , were not significantly different from the control group (98.0%) (P>0.05); the cleavage rate of the KDM6A and KDM6B double interference group was significantly lower than that of the control group (89.2% vs. 98.0%, P<0.05). In addition, The rate of blastocysts in the KDM6A interference group alone was significantly lower than that in the control group (52.7% vs. %vs.84.3%, P<0.01); at the same time, the blastocyst rate of the KDM6B-siRNA experimental group was significantly higher than that of the control group (95.1%vs.84.3%, P<0.05), as shown in Figure 24; interference was also found KDM6 can not only increase the rate of blastocysts, but also improve the quality of blastocysts, as shown in Figure 23; in order to further verify that interference with KDM6B can improve the quality of blastocysts, the blastocysts injected with KDM6B-siRNA were stained with DAPI nuclei, as shown in Figure 25 ; Statistical changes in the number of cells in blastocysts after KDM6B interference, as shown in Figure 26; the results show that the number of cells in blastocysts in the interference KDM6B group was significantly higher than that in the control group (140.7vs.116.3, P<0.01). ,

5.7.5) Reducing the expression of KDM6B helps to increase the expression of Oct4-GFP

To further explore the effect of disrupting KDM6B on embryonic development, I used oocytes from mice transgenic for GFP with the Oct4 promoter, namely OG2-oocytes, Oct4 (POU5F1), a key reprogramming factor and multiple Potency marker molecules, mainly expressed in blastocysts, are one of the gold standards for embryo quality identification. Since the OG2 transgenic mouse is a heterozygote, in order to prevent the loss of the Oct4-GFP gene during meiosis, the nested PCR method was used to prevent the loss of the Oct4-GFP gene. The donor mice with oocytes were screened, as shown in Figure 27; the embryos injected with KDM6B-siRNA were imaged by 488nm laser scanning, as shown in Figure 28, in order to obtain the dynamic expression of Oct4-GFP, KDM6B will be injected Live cell time-lapse photography of -siRNA and control-siRNA embryos, as shown in Figure 29-30, confocal and live cell results show that interfering with the expression of KDM6B can significantly increase the fluorescence signal of Oct4-GFP at the inner cell mass Intensity, thus indicating that the quality of the blastocyst has improved.

The working principle of the present invention is: the present invention detects H3K27me3 modification in mouse MII stage, 2-cell, 4-cell, 8-cell, morula, blastocyst and hatched blastocyst by immunofluorescence detection method, and then The distribution of KDM6A and KDM6B in various stages of embryonic development was detected, and it was found that H3K27me3 had dynamic changes in pre-implantation embryos, and there was no modification of H3K27me3 in 8-cell and morula stage embryos.

Previous studies have shown that H3K27me3 is an epigenetic modification that inhibits gene expression. When oocytes are activated, the fluorescence signal intensity of H3K27me3 begins to decrease. It is speculated that this decrease may be related to zygotic genome activation (ZGA); The zygote is changing from maternal regulation to zygotic regulation, which requires a large number of new gene transcriptions. Immunofluorescence and Western blot hybridization experiments show that there is a strong H3K27me3 modification in the inner cell mass of MII stage oocytes and hatched blastocysts; Studies in stem cells indicated that "the higher the degree of differentiation, the lower the expression level of H3K27me3", which is consistent with the test results in oocytes; the absence of H3K27me3 modification at the 8-cell, morula stage is consistent with previous results. Studies vary, and this variation may be due to differences in the time points at which embryos were collected.

KDM6A and KDMB6 are two enzymes that can specifically remove the methylation modification of H3K27me3 in vivo. In the current technology, the functions of KDM6A and KDM6B have only been studied in bovine pre-implantation embryos, and have not been reported in model animals and mice. The results of the present invention show that KDM6A, KDM6B are highly expressed in mouse oocytes, and the fluorescent signal of KDM6A cannot be detected in the embryos of the 8-cell stage. In addition, the present invention has also confirmed that only After double interference with KDM6A and KDM6B, H3K27me3 modification will appear in 8-cells and morula, which proves that there are only two specific demethylases, KDM6A and KDM6B, in mouse oocytes.

Through siRNA interference and western blot hybridization experiments, it was found that both KDM6A and KDM6B had functional compensation at both the mRNA level and the protein level, that is, one of them decreased and the other increased; when oocytes were injected with KDM6B-siRNA , the blastocyst rate and embryo quality of embryos were significantly better than those of the control group; not only that, the reduction of KDM6B expression was helpful for the expression of Oct4-GFP, which on the other hand showed that KDM6B was unfavorable for the development of pre-implantation embryos. However, it should be noted that the expression of KDM6A will increase after the interference of KDM6B. Whether the improvement of blastocyst development rate and embryo quality is related to the up-regulation of KDM6A expression needs further research.

The preferred implementation of this patent has been described in detail above, but this patent is not limited to the above-mentioned implementation, and within the knowledge of those of ordinary skill in the art, it can also be made without departing from the purpose of this patent. Variations.

Claims (5)

1. a kind of application processes of the H3K27me3 and its demethylase KDM6A/B in mouse nuclear transfer reconstructed embryo, its feature It is, comprises the following steps：

1) preparing experiment animal：

The donor mice (OG2) of egg mother cell when being imaged using Oct4 promoters-green fluorescent transgenic mouse as living cells；

2) MII period egg mother cells are collected：

3) preparing experiment reagent：

3.1) embryo medium：

3.2) immunofluorescence and the related main agents of Western blotting：

4% paraformaldehyde liquid, Triton X-100, phosphate buffered saline, polysorbas20, DAPI dyestuffs, antibody Oct4, antibody Nanog, antibody SSEA1, antibody Sox2, antibody H3K27me3, antibody KDM6A, antibody KDM6B, RNA extract examination Agent box, real-time fluorescence quantitative PCR；

4) quantitative primer

4.1) KDM6A, primer sequence is：

forward-TATTGGCCCAGGTGACTGTGAA

reverse-CAGATCTCCAGGTCGCTGAATAAAC

4.2) KDM6B, primer sequence is：

forward-GCTGGAGTGCTTGTTCCATGAG

reverse-GAAAGCCAATCATCACCCTTGTC

4.3) Gapdh, primer sequence is：

forward-AAAATGGTGAAGGTCGGTGTG

reverse-AATGAAGGGGTCGTTGATGG

5) experimental method

5.1) the lonely female activation of MII phases egg mother cell

5.2) siRNA microinjections

SiRNA-KDM6A, siRNA-KDM6B and siRNA- control group are diluted to respectively with the TE buffer solutions of nuclease free Liquid after dilution, is slowly injected among the kytoplasm of egg mother cell by 40mM using micromanipulation arm by injection needle, note Egg mother cell room temperature after penetrating is transferred to KSOM nutrient solutions after recovering 15min, is placed in 2h in 37 DEG C of incubators；

5.3) real-time quantitative PCR, advises according to quantitative specification, is tested；

5.4) immunofluorescence dyeing

5.4.1) embryo fixes：Embryo is put into the paraformaldehyde fixer that mass fraction is 4%, room temperature 60min or 4 DEG C of bars Fixation is stayed overnight under part；

5.4.2 it is) penetrating：Embryo after fixation is cleaned one time with 1ml phosphate buffered salines, moving into mass fraction is 0.5% Triton X-100 solution room temperature treatment 15min；

5.4.3) close：It is 1% that embryo is put into containing mass fraction into the Triton X-100 and mass fraction that are 0.2% Bovine serum albumin solution in room temperature closing 30min；

5.4.4) antibody incubation：Embryo is moved into 37 DEG C of incubation 1h in the antibody crossed through diluted, dilution is containing quality point The bovine serum albumin(BSA) that the polysorbas20 and mass fraction that number is 1% are 1%, 3 are rinsed after incubation with phosphate buffered saline Secondary, each 5min adds the antibody-solutions of confining liquid dilution, confining liquid is containing the polyethylene glycol octyl group benzene that mass fraction is 0.2% Base ether and the bovine serum albumin solution that mass fraction is 1%, room temperature lucifuge are kept away after being incubated 1h with phosphate buffered saline Light is rinsed 3 times, each 5min；

5.4.5) DAPI dyeing：By embryo's room temperature lucifuge dye 15min, with phosphate buffered saline lucifuge rinse 3 times, often Secondary 5min；

5.4.6) mounting：The anti-quenching medium of drop of plus one moves into anti-essence on slide, by the embryo after dyeing and gone out in agent, covers lid glass Mountant mounting after piece；

5.4.7) co-focusing imaging：Image is gathered using Nikon A1R Laser Scanning Confocal Microscopes；

5.5) living cells is imaged：Egg mother cell after microinjection siRNA, after lonely female activation 6h, is incubated at Nikon Ti-E Install living cells imaging system additional, and use 488nm laser scannings, be imaged by Nikon A1R camera scannings；

5.6) data statistics and analysis.

2. H3K27me3 according to claim 1 and its demethylase KDM6A/B is in mouse nuclear transfer reconstructed embryo Application process, it is characterised in that in the step 1, the mouse is purchased from University of the Inner Mongol's Animal Experimental Study center, donor mouse Raise in SPF grades of 22~24 DEG C of Animal Houses, relative humidity 50~60%, Animal House light application time is early 8 points to late 8 points daily.

3. H3K27me3 according to claim 1 and its demethylase KDM6A/B is in mouse nuclear transfer reconstructed embryo Application process, it is characterised in that in the step 3.1, the compound method of embryo medium is：

1) the dense liquid storages of HCZB：500mL；Constituent is：Ultra-pure water 495ml, sodium chloride 2380mg, potassium chloride 180mg, 7 water sulphur Sour magnesium 145mg, disodium ethylene diamine tetraacetate 20mg, sodium lactate 2.65mL, glucose 500mg, potassium dihydrogen phosphate 80mg；

2) the dense liquid storages of 10ml calcium chloride 100X：It is formulated by 0.2g calcium chloride dihydrates and 10ml ultra-pure waters；

3) the dense liquid storages of 10ml strontium chlorides 100X：It is formulated by the water strontium chlorides of 2.666g six and 10ml ultra-pure waters；

4) the dense liquid storages of 20ml glutamine 100X：It is formulated by 0.294g glutamine and 20ml without calcium CZB solution；

5) cytochalasin B solution：By cytochalasin B with 1mg/ml proportioning be dissolved in dimethyl sulphoxide solution prepare and Into；

6) polyvinylpyrrolidonesolution solution：

Mass fraction is 10% PVP solution：It is formulated by 1g PVP and 10ml HCZB；

Mass fraction is 3% PVP solution：It is formulated by 0.3g PVP and 10ml HCZB；

7) hyaluronidase solution：It is formulated by 20ml M2 nutrient solutions and 100mg hyaluronidases.

4. H3K27me3 according to claim 1 and its demethylase KDM6A/B is in mouse nuclear transfer reconstructed embryo Application process, it is characterised in that in the step 5.2, the compound method of KSOM nutrient solutions is：Per 100mLKSOM-AA embryos training 0.38mg containing disodium EDTA in nutrient solution, sodium chloride 559.5mg, potassium chloride 18.5mg, potassium dihydrogen phosphate 4.75mg, 7 water magnesium sulfate 4.95mg, lactylate salt solution 0.174ml, glucose 3.60mg, sodium acid carbonate 210.0mg, glutamine 14.5mg, Sodium Pyruvate 2.2mg, dual anti-1 6.3mg, dual anti-2 5.0mg, essential amino acid 1.0ml, nonessential amino acid 0.5ml；Wherein disodium EDTA is preferentially added dropwise, and 100mg bovine serum albumin(BSA)s are added into 100mLKSOM-AA embryos In tire nutrient solution, gently mixing makes bovine serum albumin(BSA) slow mechanism dissolved, should not be aggressively shaken liquid and be led with avoiding generation foam Cause protein denaturation；With in 0.22 μm of syringe filter nutrient solution to sterile plastic tube, solution is stored in 4 DEG C, guarantor Deposit the time 1~2 week.

5. H3K27me3 according to claim 1 and its demethylase KDM6A/B is in mouse nuclear transfer reconstructed embryo Application process, it is characterised in that in the step 5.6, data statistics is with analysis method using SPSS10.0 statistics softwares point Analysis is handled, P<0.05 is significant difference, P<0.01 is that difference is extremely notable.