Construction method and application of pig KDM5B gene knockout cell

Abstract

The invention discloses a construction method and application of a cell with a pig KDM5B gene knocked out, the construction method of the cell with the pig KDM5B gene knocked out comprises the following steps: S1, designing guide RNA (sgRNA): designing a sgRNA sequence for subsequent targeting aiming at a coding region of the pig KDM5B gene; s2, constructing a carrier: selecting sgRNA to construct a pX330-gRNA carrier; s3, cell transfection and genotype identification: co-transfecting the vector constructed in S2 and pCAG-GFP plasmids into porcine fetal fibroblasts, carrying out single cell sorting through a flow cell sorter according to the expression condition of GFP, and culturing to obtain single cell clones. According to the method disclosed by the invention, the donor cell with the pig KDM5B gene knocked out is obtained, and the influence of the KDM5B gene on cloning efficiency and a specific action mechanism of the KDM5B gene on epigenetics are explored by comparing the donor cell with a wild donor cell. The method provided by the invention is helpful for remarkably improving the in-vitro developmental ability of the pig cloned embryo and further improving the cloning efficiency.

Description

Technical Field

[0001] The invention relates to the fields of biotechnology and fermentation technology, and in particular to a method for constructing a cell for knocking out a pig KDM5B gene and an application thereof. Background Art

[0002] In animal husbandry production, pigs are important agricultural economic animals and provide the main source of meat worldwide. In China, pork consumption accounts for about 65% of the total meat consumption in national life. Improving the growth rate, reproductive capacity, meat quality traits, disease resistance and feed conversion rate of pigs is an urgent need for variety breeding in China's pig farming industry. Conventional breeding technology has made remarkable achievements in realizing the utilization of hybrid advantages of livestock and poultry and improving economic traits, but its long selection cycle, slow genetic progress, and difficulty in hybridization between different species have always been the "technical bottleneck" for breeding new varieties. In modern animal molecular breeding, the degree of screening and improvement assisted by molecular marker technology is still limited by the genetic diversity and gene resources within the variety. Genetic modification of pigs through gene editing technology can effectively circumvent the above "technical bottleneck", break the natural barrier of reproductive isolation between species, and enable breeding work to make full use of all possible genetic variations, even genes from lower organisms or plants, thereby greatly shortening the generation interval, greatly improving the progress speed and richness of livestock and poultry improvement, and at the same time creating some unconventional products according to people's needs.

[0003] At present, in the field of biomedicine, pigs are ideal large animal models for human diseases. On the one hand, pigs are distantly related to humans and do not have the ethical issues involved in primate model animals such as monkeys and gorillas; on the other hand, the organ size, anatomical structure, metabolic characteristics and physiological functions of pigs are much more similar to those of humans than rodent model animals such as mice and rats. In addition, the operability of clinical medical technologies such as surgical surgery and imaging diagnosis makes pigs suitable animals for the preparation of animal models of human diseases such as cardiovascular diseases, neurodegenerative diseases, ophthalmic diseases, carbohydrate and lipid metabolic diseases, immunodeficiency, cancer and xenotransplantation. Therefore, the efficient construction of pig models of human diseases and xenotransplantation using genome editing technology will be of great significance to the field of biomedicine.

[0004] Due to the lack of pig embryonic stem cell lines, somatic cell nuclear transfer (SCNT) technology is the main way to prepare cloned pigs and genome-edited model pigs. SCNT technology is an effective solution for breeding new pig breeds and building human disease models. However, no technology can be perfect and has its drawbacks. SCNT technology also has its unavoidable "technical bottlenecks". The cloning efficiency of almost all mammals is less than 5%. How to improve the SCNT efficiency of mammals has always been the pursuit of scientific researchers. A large number of studies accumulated over decades have shown that incomplete epigenetic reprogramming is an important reason affecting the efficiency of mammalian SCNT.

[0005] Early mammalian embryonic development is a complex biological process. To ensure the completion of various developmental events, the coordinated action of various transcription factors and epigenetic modifications is required. These important developmental events include fertilization, zygotic genome activation (ZGA), and the first cell fate determination. Epigenetic modification is essential for early mammalian embryonic development, and somatic cell nuclear transfer embryos show a variety of epigenetic modification defects, including histone acetylation, histone methylation, DNA methylation, and X chromosome inactivation disorder. Studies on improving somatic cell reprogramming by changing epigenetic modifications suggest that manipulating the epigenetic modification sites of somatic cells and changing the chromosome state and structure of somatic cells should be the most effective strategy to improve somatic cell reprogramming.

[0006] As mentioned above, embryonic zygotic genome activation is the first important node of embryonic development after fertilization, which will have a significant impact on the entire embryonic development event. Studies have found that the zygotic genome activation level of mammalian SCNT embryos is significantly lower than that of normal embryos, and the activation time is also significantly delayed. Zygotic genome activation is inextricably linked to histone modification, suggesting that we can rescue the abnormal zygotic genome activation of SCNT embryos by improving histone modification.

[0007] Therefore, the present invention explores the effect of KDM5B on pig cloned embryos on the basis of the above. This study will further detect the effect of KDM5B functional deficiency on the developmental ability of cloned embryos and comprehensively analyze its mechanism of action. Summary of the invention

[0008] The present invention aims to solve the above problems and provides a method for constructing cells with knockout porcine KDM5B gene and its application. The specific technical scheme is as follows: In a first aspect, the present invention provides a method for constructing a cell in which the porcine KDM5B gene is knocked out, the method comprising the following steps: S1: Design guide RNA (sgRNA): Design the sgRNA sequence for subsequent targeting against the coding region of the porcine KDM5B gene (Ensembl: ENSSSCG00000010928); S2: Vector construction: Select sgRNA to construct pX330-gRNA vector; S3: Cell transfection and genotype identification: The vector constructed in S2 was co-transfected with the pCAG-GFP plasmid into porcine fetal fibroblasts. Single cells were sorted by flow cytometry based on the expression of GFP, and single cell clones were obtained by culture.

[0009] Furthermore, the specific steps of constructing the vector in step S2 are as follows: S21: linearize the pX330 vector with BbsI enzyme and perform gel excision and recovery; S22: Synthesis of double-stranded sgRNA: Take equal amounts of the upstream strand and the downstream strand, mix them, and anneal them to form a double strand; S23: sgRNA is connected to the linearized pX330 vector, and the ligation product is then transformed, and the single clone with the correct sequencing result is subjected to plasmid extraction.

[0010] In a second aspect, the present invention provides porcine KDM5B gene knockout cells prepared by the method described in the first aspect.

[0011] In a third aspect, the present invention provides a method for constructing a KDM5B gene knockout pig SCNT reconstructed embryo based on the pig KDM5B gene knockout cells described in the second aspect, the method comprising the following steps: S1: Place the oocyte in the operating solution on the stage of the inverted microscope of the micromanipulator, use the micromanipulator needle to aspirate 1 / 10 of the cytoplasm around the first polar body to remove the nucleus of the oocyte. The enucleated oocyte is stored in the operating solution and placed in a 38°C constant temperature incubator with humidity maintained. After all the enucleations are completed, the dead oocytes and those with poor morphology are discarded; S2: When injecting cells, select pig fetal fibroblasts with regular shape, uniform size, uniform cytoplasm and vigorous vitality and inject them into the perivitelline space of the enucleated oocyte. Then, place the injected pig oocyte in the embryo culture droplet PZM3 to recover for about 0.5 h. S3: The recovered oocytes that have been enucleated and injected with nuclei are transferred in batches to the fusion solution for washing and equilibration for 2-3 minutes, and then transferred to the fusion tank filled with fusion solution. The position of the reconstructed embryo is adjusted so that the contact surface between the donor cell and the recipient oocyte is perpendicular to the direction of the electric field. Then, two repeated direct current pulses of 1.2 kV / cm, 30 μs, and 1 s interval are applied with an electric fusion instrument to induce fusion and activate at the same time. The electroshocked embryos are transferred to the porcine embryo culture medium PZM3 for 30 minutes and then the fusion status is determined; S4: The fused reconstructed embryos were transferred into embryo culture droplets PZM3 that had been pre-equilibrated for more than 4 hours and continued to be cultured at 39°C, 5% CO2 and saturated humidity. The cleavage rate was recorded after 48 hours of observation and the blastocyst rate was recorded after 144 hours of observation.

[0012] In a fourth aspect, the present invention provides a pig KDM5B gene knockout pig SCNT reconstructed embryo obtained by the method described in the third aspect.

[0013] Furthermore, the present invention also provides the use of porcine KDM5B gene knockout cells as described in the second aspect in porcine SCNT reprogramming.

[0014] The present invention has the following advantages / benefits: 1. The present invention provides a method for constructing cells with pig KDM5B gene knocked out, and obtains donor cells with pig KDM5B gene knocked out by this method. By comparing the cells with wild-type donor cells, the effects of KDM5B gene on cloning efficiency and the specific mechanism of epigenetic effects are explored.

[0015] 2. The experimental data of the present invention show that KDM5B - / - Compared with the wild type, the blastocyst rate and the total number of cells in the blastocyst were significantly improved; KDM5B knockout cell nuclear transplantation reprogramming, the knockout of the KDM5B gene increased the looseness of the cell nuclear chromatin; reduced the expression of HOX genes in the early 4-cell stage - that is, the early activation of the zygotic genome; increased the expression of gene expression and regulation, RNA synthesis / metabolism and regulation, cell metabolism and other life activity-related signal pathways in somatic cell nuclear transplanted embryos.

[0016] 3. The method provided by the present invention helps to significantly improve the in vitro development ability of pig cloned embryos and further improve the efficiency of pig cloning. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0018] Figure 1 :The effect of knocking out KDM5B in donor cells of the present invention on SCNT of porcine oocytes; A is the rate of reconstructed embryos and embryo blastocysts, B is the total number of cells contained in the blastocysts, and C is the morphological observation of embryo blastocysts on the sixth day; Figure 2 : The effect of KDM5B deficiency on histone methylation and chromatin looseness in fibroblasts; A is the detection of H3K4me3 and H3K27me3 protein levels, B is the H3K4me3 and H3K27me3 cell immunofluorescence detection, and C is the MNase (micrococcal nuclease) sensitivity analysis; Figure 3 : GO functional enrichment analysis of differentially expressed genes in WT-NT and KO-NT groups of 4-cell embryos of the present invention; wherein A is the heat map clustering of differentially expressed genes, B is the GO functional enrichment analysis of down-regulated genes in KO-NT samples, and C is the GO functional enrichment analysis of up-regulated genes in KO-NT samples; Figure 4 : The present invention analyzes the positive and negative regulation genes of KDM5B in the porcine somatic cell nuclear transfer embryo system; wherein A is the intersection of the genes that are highly expressed and lowly expressed in WT-NT and KO-NT samples compared with IVF-derived embryos, B is the number of KDM5B positive and negative regulation genes in the porcine somatic cell nuclear transfer embryo system, and C is the GO functional enrichment analysis of the KDM5B positive and negative regulation genes in the porcine somatic cell nuclear transfer embryo system. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0020] 1. Materials and Methods 1.1 Experimental Materials The ovaries of young sows were all sourced from local slaughterhouses, and the semen was taken from local breeding farms; the 35-day-old fetuses were all sourced from the experimental pig farm of Qingdao Agricultural University.

[0021] 1.2 Reagents The components of the reagents used in the in vitro maturation of porcine oocytes, including PVA-TL-HEPES (oocyte washing solution), IVM Stock B (porcine oocyte in vitro maturation solution storage solution), IVM (porcine oocyte in vitro maturation solution working solution), oocyte operating solution, hyaluronidase (degranulated cell solution), PZM3 (porcine embryo in vitro culture solution), FM (fusion solution), sperm wash (sperm washing solution), 60% percoll (sperm processing solution), and mTBM (porcine in vitro fertilization solution) were purchased from Sigma unless otherwise specified.

[0022] DPBS, digestion solution and culture medium were used during the establishment and culture of porcine primary fibroblasts.

[0023] pXT7-hCas9 was obtained from the Chinese Zebrafish Research Center; MEGAscript™ T7 Transcription Kit (Ambion); RNA purification kit (Tiangen Biochemical); In vitro Transcription T7 Kit (Takaro); mirVana™ miRNA Isolation Kit (Ambion); restriction endonucleases (NEB); Cas9 protein (NEB); primer synthesis and nucleic acid sequencing were all provided by Invitrogen.

[0024] Micro-sample RNA extraction kit QiagenAllPrep DNA / RNA Micro Kit (Invitrogen, California, MA); reverse transcription kit TIANscript RT Kit (TIAGEN, Beijing, China); fluorescence quantitative PCR kit TaKaRa SYBR® Premix Ex Taq TM (TaKaRa, Tokyo, Japan); immunofluorescence test requires preparation of PBS, fixative, permeabilization solution, elution solution and blocking solution, etc.; immunoblotting uses polyacrylamide gradient gel, electrophoresis buffer, transfer solution, etc.

[0025] Main reagent formula: The formula of PVA-TL-HEPES: NaCl (6.663 g), KCl (0.237 g), NaHCO 3(0.168 g), NaH 2 PO 4 (0.041g), NaLactate (1.868mL), MgCl 2 6H 2 O (0.102g), Hepes (2.383g), PenicillinG (0.065g), PhenolRed (0.010g), CaCl 2 .2H 2 O (0.294g), Polyvinylalcohol (0.100g), Sorbitol (2.186g), Gentamicin (0.025g), Sodiumpyruvate (0.022g), MilliQH 2 O (1000 mL); Formula of porcine oocyte maturation solution: PVA (5%) (2mL), NaHCO 3 (0.25g), Gentamicin (100uL), D-glucose (0.0548g), Sodiumpyruvate (0.01g), TCM-199 (0.95g), MilliQH 2 O (98mL); Formula of porcine oocyte operation fluid: TCM-199 (9.500g), NaHCO 3 (0.050g), Hepes (0.750g), PenicillinG (0.050g), Streptomycin (0.060g), NaCl (1.755g), BSA (3.00g), MilliQH 2 O (1000mL); Hyaluronidase solution formula: Hyaluronidase (100mg), Mannitol (6g), BSA (0.001g), PVA-TL-Hepes (5mL), MilliQH 2 O (95mL), Fusion solution formula: Mannitol (0.3M), CaCl 2 .2H 2 O (1.0 mM), MgCl 2 6H 2 O (0.1mM), Hepes (0.5mM); Formula of pig embryo culture medium stock solution: NaCl (6.311g), KCl (0.7455g), KH 2 PO 4 (0.0476 g), MgSO 4 ( 0.0986 g ), NaHCO 3 (2.106g), Gentamicin (10 g), SigmaH 2 O (1000mL); PBS formula: NaCl (8 g), KCl (0.20 g), Na 2 HPO 4 ( 1.15 g ),KH 2 PO 4 ( 0.20 g ),MilliQH 2 O (1000mL); Polyacrylamide gradient gel L solution (4%): 30% polyacrylamide stock solution (5.8mL), 1.5MTris (pH8.8) (11.3mL), ddH 2 O (26 mL), 10% APS (450 μL), TEMED (22.5 μL), total 45 mL; Polyacrylamide gradient gel H solution (20%): 30% polyacrylamide stock solution (30 mL), 1.5 M Tris (pH 8.8) (11.3 mL), ddH 2 O (2.7mL), 10% APS (450μL), TEMED (22.5μL), total 45mL.

[0026] 1.3 Instruments Electric blast drying oven (DHG-9245A), electric constant temperature water bath (DK-8D), and bacterial incubator (DHP-9162) were all purchased from Shanghai Yiheng Co., Ltd.; nucleic acid concentration meter (Nano Drop 2000), cell culture incubator (HERA cell150), ultra-low temperature refrigerator (FORMA 900 series), normal temperature high-speed centrifuge (Eppendorf Centrifuge 5424), low-temperature high-speed centrifuge (Eppendorf Centrifuge 5424 R), and embryo incubator (HERA cell 150i) were all purchased from Thermo Company, USA; inverted microscope (Eclipse TS 100), stereoscope (SMZ1000), and micromanipulator were purchased from Nikon Company, Japan; transfer instrument power supply (Powerpac HC), protein electrophoresis instrument, and protein transfer instrument (221BR44245) were all purchased from BIO-RAD Company, USA; gene amplification PCR The electrophoresis instrument (EDC810) was purchased from Beijing Dongshenglong Company. The nucleic acid gel imaging and analysis system (Tanon-1600) was purchased from Shanghai Tianneng Company. The electrophoresis power supply (DYY-6C) was purchased from Beijing Liuyi Company. The digital balance (ET214S) was purchased from Beijing Sartorius Company. The constant temperature metal bath (A1901115) was purchased from Beijing Dingguo Company. The Odyssey infrared scanning imaging system (LI-COR) was purchased from Gene Company. The clean bench (DL-CJ-IND) was purchased from Beijing Donglian Company. The ultraviolet spectrophotometer (721 model) was purchased from Shanghai Jinghua Company. The pH meter (PH211) was purchased from HANNA Company, USA. The decolorization shaker (TS-8S) was purchased from Jiangsu Qilin Bell Company. The magnetic stirrer (78-1) was purchased from Jiangsu Ronghua Company; the fusion instrument Electro-cell Manipulator 2000 was purchased from BTX Company, USA 1.4 Experimental methods 1.4.1 Preparation of primary porcine fetal fibroblasts Porcine fetal fibroblasts were obtained from fetuses at 35 days of gestation. After anesthetizing the sows at 35 days of gestation, the uterus was removed and quickly transferred to a clean room. The fetal membranes and placenta were carefully peeled off with scissors and forceps. The separated fetus was placed in 1×DPBS containing 40ug / mL gentamicin and washed several times until there was no blood. In the clean bench, the head, tail and limbs were carefully removed with scissors, and the abdominal cavity was separated with forceps to remove the internal organs. The remaining trunk was rinsed several times with DPBS containing antibiotics until it was clean. After removing the head, tail and limbs, it was placed in a 3cm sterile culture dish and the tissue was cut into pieces with scissors. The tissue blocks were cut as much as possible to be less than 1 mm in size. 3The chopped tissue pieces were resuspended in DPBS and added to a 15 mL centrifuge tube. After standing for a few minutes, the supernatant was discarded and the tissue pieces were transferred to the cell culture medium containing collagenase (2000 U / mL) and DNaseI (25 KU / mL). 2 (100% humidity, 39°C, 5% CO 2 After digestion, centrifuge at 500×g for 5 min, discard the supernatant, resuspend in cell culture medium, and transfer to 75cm 2 Culture and freeze after confluence.

[0027] 1.4.2 In vitro maturation of porcine oocytes and production of embryos using somatic cell nuclear transfer The ovaries of young sows obtained from the local slaughterhouse were transported back to the laboratory, and the follicles were aspirated. After the obtained follicular fluid was repeatedly allowed to settle, the cumulus-oocyte complex with uniform cytoplasm, more than three layers of cumulus cells and tight packaging was picked up under a stereomicroscope, added to the porcine oocyte maturation culture medium, and placed in CO 2 In vitro maturation culture was carried out in an incubator. After 42-44 hours, mature oocytes with one polar body and uniform cytoplasm were selected under a stereomicroscope for subsequent experiments.

[0028] After aspirating 1 / 10 of the cytoplasm around the polar body using a micromanipulation system on an inverted microscope, the donor cells were injected into the perivitelline space, electrofused, and added to the pig early embryo culture medium. 2 The cells were cultured in vitro for maturation in an incubator, cleavage was observed for 24 hours, and the blastocyst rate was counted on day 6.

[0029] 1.4.3 Immunofluorescence of embryos and cells After using 4% paraformaldehyde to fix porcine oocytes, somatic cells and embryos of various periods at room temperature for 30 minutes, the membrane was permeabilized at room temperature for 20 minutes, and then blocked with blocking solution at room temperature for 1 hour. After blocking, the samples were incubated overnight at 4°C with primary antibodies. The samples incubated overnight with primary antibodies were washed three times with elution solution, each for 5 minutes. After elution, the samples were combined with secondary antibodies at room temperature for 1 hour, and the light was blocked and eluted three times, each for 5 minutes. Then, the nuclei were stained with hoechst (blue) or PI (red), and the hoechst staining was 15 minutes. The slides were directly sealed without elution. PI staining was 8 minutes. After elution with elution solution three times, the slides were sealed and stored.

[0030] 1.4.4 Embryo transcriptome sequencing Early 4-cell embryos derived from wild-type donor cells and KDM5B (Ensembl: ENSSSCG00000010928) knockout donor cells were collected and delivered to sequencing companies for transcriptome sequencing and analysis.

[0031] 1.4.5 Cell Western Blot Sample lysis: Take about 200 pig embryos (the number of cells is not less than 10 4 ) Add loading buffer in proportion so that the volume does not exceed 20 μL, lyse on ice for 10 min, and heat the sample at 95°C for 3-5 min to denature.

[0032] Immunoblotting: Add the prepared protein sample to the spotting well to prevent the sample from overflowing and contaminating other spotting wells. Adjust the voltage according to the experimental purpose and monitor the electrophoresis operation according to the position of bromophenol blue. Pour the transfer solution into the glass dish, cut the filter paper and nitrocellulose membrane of appropriate size in turn, and soak them in the transfer solution; turn on the semi-dry transfer membrane instrument, and put the filter paper, cellulose membrane, PAGE gel after electrophoresis, and filter paper from bottom to top in turn, try to avoid bubbles, install the transfer membrane instrument, and transfer the membrane with a voltage of 25V and a current of 16mA. The transfer time depends on the size of the protein, generally about 1h. After transfer, place the membrane in 5% skim milk or 5% BSA (prepared with 1×PBS) for blocking for 30 minutes; transfer the membrane to 5% skim milk containing primary antibody and incubate overnight at 4°C; on the next day, wash the membrane 3 times with 1×PBS, 5 minutes each time; transfer the membrane to the secondary antibody dilution solution containing secondary antibody and incubate at room temperature for 1 hour; wash the membrane 3 times with 1×PBS, 10 minutes each time; the secondary antibody used in the experiment is fluorescent secondary antibody, and the membrane can be scanned with a membrane scanner to detect protein signals.

[0033] 1.5 Statistical analysis All experiments involved in the study were repeated three times. Quantitative PCR, immunofluorescence data, blastocyst cell number, and apoptotic cell ratio were analyzed using Student's t-test; the differences in cleavage rate and blastocyst rate were analyzed using chi-square test. P values ​​were less than 0.05 (*) or 0.01 (**).

[0034] Example 1: Study on the effect of KDM5B knockout in donor cells on pig SCNT reprogramming The effect of KDM5B on porcine somatic cell nuclear transfer reprogramming. The screened double-allelic knockout cell lines were used as donors to construct porcine SCNT reconstructed embryos, and the untargeted wild-type cell lines screened during the same period were used as donors to construct porcine SCNT reconstructed embryos as controls.

[0035] The results of reconstructed embryonic development are shown in Tables 1 and Figure 1As shown: Knocking out KDM5B in donor cells had no significant effect on the cleavage rate of reconstructed embryos (90.9% Vs. 92.1%, P>0.05), but it could significantly increase the blastocyst rate of fifth-day embryos (11.7% Vs. 23.5%, P<0.01), the blastocyst rate of sixth-day embryos (20.3% Vs. 40.4%, P<0.01), and the total number of cells contained in the blastocyst also increased significantly (35.06±2.54 Vs. 42.84±3.21, P<0.01).

[0036] These results indicate that knocking out donor cell-derived KDM5B can significantly improve the efficiency of pig SCNT reprogramming.

[0037] Table 1 Developmental capacity of wild-type SCNT embryos and KDM5B knockout SCNT embryos

[0038] Example 2: KDM5B can regulate porcine SCNT reprogramming by regulating the chromatin loosening, cell proliferation and cell cycle of donor cells.

[0039] In order to study the potential mechanism by which KDM5B knockout in donor cells promotes pig somatic cell nuclear transfer reprogramming, the abundance of histone modifications H3K4me3 and H3K27me3 was first detected at the cellular level. Histone modifications H3K4me3 and H3K27me3 play an important role in early mammalian embryonic development. Histone modification H3K4me3 is associated with transcriptional activation, while histone modification H3K27me3 is associated with transcriptional repression. WB and immunofluorescence results showed that the histone modification H3K4me3 in the KDM5B knockout cell line was significantly increased ( Figure 2 -A,2-B), while the level of histone modification H3K27me3 was significantly reduced ( Figure 2 -A, 2-B), suggesting that there is a negative correlation between the transcriptional activation-related histone mark H3K4me3 and the transcriptional repression-related histone mark H3K27me3 based on the KDM5B gene in porcine fetal fibroblasts.

[0040] The abundance of histone modifications H3K4me3 and H3K27me3 is often closely related to chromosome structure. In order to study the effect of KDM5B functional loss on chromosome structure in porcine fetal fibroblasts, the micrococcal nuclease sensitivity test was used to detect the expression of KDM5B in wild-type cells and KDM5B - / - The results showed that knocking out KDM5B could increase the looseness of nuclear chromatin ( Figure 2 -C).

[0041] Example 3: Single-cell transcriptome sequencing technology was used to study the effect of donor-derived KDM5B on pig SCNT zygotic genome activation.

[0042] KDM5B deficiency in donor cells reduced the expression of HOX family genes in pig SCNT embryos at the early 4-cell stage, i.e., early zygotic genome activation.

[0043] The results of differential analysis of transcriptome samples of early 4-cell embryos derived from wild-type donor cells and KDM5B knockout donor cells showed that a total of 387 differentially expressed genes were detected (P < 0.01, Log2 (FC) > 1.5), of which 280 genes were upregulated in early 4-cell embryos derived from KDM5B knockout donor cells, while the other 107 genes were highly expressed in embryos derived from wild-type donor cells.

[0044] In order to understand the functions of the 107 genes whose expression decreased in early embryos due to the loss of KDM5B in donor cells, we performed GO functional gene enrichment on the above 107 genes. The results showed that the above genes were enriched in signal pathways closely related to embryonic skeletal system morphogenesis, embryonic organ morphogenesis, embryonic morphogenesis, and organ differentiation in the middle and late stages of embryonic development.

[0045] We found that most of the genes involved in the above signaling pathways closely related to organ differentiation in the middle and late stages of embryonic development belong to the HOX family genes, such as HOXA5, HOXB6, HOXA11, HOXB5 and HOXA2 ( Figure 3 ). Previous studies have found that high expression of HOX family genes in pig preimplantation embryos is detrimental to early embryonic development. Reducing the expression of HOX family genes in somatic cell nuclear transfer-derived embryos in the early 4-cell stage, i.e., early zygotic genome activation, may be one of the potential mechanisms by which KDM5B functional deficiency in donor cells improves the developmental capacity of pig somatic cell nuclear transfer embryos.

[0046] Example 4: KDM5B deficiency in donor cells can increase the gene expression and regulation, RNA biosynthesis / metabolism and regulation, cell metabolism and other life activity-related signal pathway gene expression in somatic cell nuclear transplanted embryos.

[0047] In addition, in order to further understand the effect of KDM5B functional loss in donor cells on the developmental signaling network of pig somatic cell nuclear transfer embryos and to explain its potential mechanism for promoting pig somatic cell nuclear transfer reprogramming, we took the intersection of genes that were highly expressed and lowly expressed in WT-NT and KO-NT samples compared with IVF-derived embryos. (WT-NT: somatic cell nuclear transfer using wild-type pig fibroblasts as donor cells; KO-NT: somatic cell nuclear transfer using KDM5B- / - pig fibroblasts as donor cells) We found that the number of genes with significantly lower expression in WT-NT and KO-NT samples compared with IVF samples was 688 and 375, respectively, and there was no overlap between the two, indicating that knocking out the KDM5B gene in donor cells significantly increased the expression of 688 genes that were low in wild-type NT embryos. These 688 genes may be directly or indirectly negatively regulated by KDM5B. In KO-NT embryos, the 375 genes with significantly lower expression may be directly or indirectly positively regulated by KDM5B ( Figure 4 -A, Figure 4 -B).

[0048] We found that 304 of the 479 genes that were significantly more highly expressed in IVF embryos than in NT-WT embryos were restored by knocking out KDM5B in donor cells. Therefore, the above 304 genes may also be indirectly or directly negatively regulated by KDM5B. In addition, we also found that 385 genes that were significantly more highly expressed in IVF embryos than in NT-KO embryos, excluding 175 genes that existed in both NT-derived embryos (WT-NT and KO-NT), the remaining 210 genes may be directly or indirectly positively regulated by KDM5B ( Figure 4 -A, Figure 4 -B).

[0049] By comparing the transcriptomes of IVF-derived embryos and NT-derived embryos, and taking the intersection of genes with increased expression and genes with decreased expression, we found that during pig embryo development, 898 genes were overexpressed due to the loss of KDM5B function in donor cells; at the same time, 679 genes were decreased in expression due to the loss of KDM5B function in donor cells. GO functional enrichment was performed on the 898 genes whose expression was upregulated due to the loss of KDM5B function in donor cells and the 679 genes whose expression was downregulated. Figure 4 -C).

[0050] The analysis results showed that 898 genes whose expression was upregulated due to loss of KDM5B function in donor cells were mostly enriched in signal networks related to activation of embryonic biological activities such as biosynthesis, gene expression, and RNA metabolism; and very interestingly, 679 genes whose expression was downregulated due to loss of KDM5B function in donor cells were mostly enriched in signal networks related to inhibition of embryonic biological activities such as inhibition of biosynthesis, inhibition of gene expression, and inhibition of RNA metabolism. This further proves that the loss of KDM5B in donor cells can increase the expression of genes in signal pathways related to life activities such as gene expression and regulation, RNA biosynthesis / metabolism and regulation, and cell metabolism in somatic cell nuclear transfer embryos, and improve the abnormal expression of the above-mentioned genes related to life activities in pig somatic cell nuclear transfer embryos.

[0051] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A method for constructing a cell with knockout of pig KDM5B gene, It is characterized in that The method comprises the following steps: S1: Design guide RNA (sgRNA): Design the sgRNA sequence for subsequent targeting against the coding region of the porcine KDM5B gene; S2: Vector construction: Select sgRNA to construct pX330-gRNA vector; S3: Cell transfection and genotype identification: The vector constructed in S2 was co-transfected with the pCAG-GFP plasmid into porcine fetal fibroblasts. Single cells were sorted by flow cytometry based on the expression of GFP, and single cell clones were obtained by culture.

2. A method for constructing a cell with pig KDM5B gene knocked out according to claim 1, It is characterized in that The specific steps of constructing the vector in step S2 are as follows: S21: linearize the pX330 vector with BbsI enzyme and perform gel excision and recovery; S22: Synthesis of double-stranded sgRNA: Take equal amounts of the upstream strand and the downstream strand, mix them, and anneal them to form a double strand; S23: sgRNA is connected to the linearized pX330 vector, and the ligation product is then transformed, and the single clone with the correct sequencing result is subjected to plasmid extraction.

3. The porcine KDM5B gene knockout cells obtained according to the method of claim 1.

4. A method for constructing KDM5B gene knockout pig SCNT reconstructed embryos based on the pig KDM5B gene knockout cells as claimed in claim 3, It is characterized in that The method comprises the following steps: S1: Place the oocyte in the operating solution on the stage of the inverted microscope of the micromanipulator, use the micromanipulator needle to aspirate 1 / 10 of the cytoplasm around the first polar body to remove the nucleus of the oocyte. The enucleated oocyte is stored in the operating solution and placed in a 38°C constant temperature incubator with humidity maintained. After all the enucleations are completed, the dead oocytes and those with poor morphology are discarded; S2: When injecting cells, select pig fetal fibroblasts with regular shape, uniform size, uniform cytoplasm and vigorous vitality and inject them into the perivitelline space of the enucleated oocyte. Then, place the injected pig oocyte in the embryo culture droplet PZM3 to recover for about 0.5 h. S3: The recovered oocytes that have been enucleated and injected with nuclei are transferred in batches to the fusion solution for washing and equilibration for 2-3 minutes, and then transferred to the fusion tank filled with fusion solution. The position of the reconstructed embryo is adjusted so that the contact surface between the donor cell and the recipient oocyte is perpendicular to the direction of the electric field. Then, two repeated direct current pulses of 1.2 kV / cm, 30 μs, and 1 s interval are applied with an electric fusion instrument to induce fusion and activate at the same time. The electroshocked embryos are transferred to the porcine embryo culture medium PZM3 for 30 minutes and then the fusion status is determined; S4: The fused reconstructed embryos were transferred into embryo culture droplets PZM3 that had been pre-equilibrated for more than 4 hours and continued to be cultured at 39°C, 5% CO2 and saturated humidity. The cleavage rate was recorded after 48 hours of observation and the blastocyst rate was recorded after 144 hours of observation.

5. The porcine KDM5B gene knockout pig SCNT reconstructed embryo obtained according to the method of claim 4. 6 . Use of the porcine KDM5B gene knockout cells as claimed in claim 3 in porcine SCNT reprogramming.

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