Primitive-state-like bovine embryonic stem cell line as well as induction method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a primitive-like bovine embryonic stem cell line as well as an induction method and application thereof, and the bovine embryonic stem cell line overexpresses a gene JUP. The primitive-like pluripotent stem cell line (bESCs) is obtained through gene JUP overexpression, a part of important primitive marker genes are obtained through screening, and the bovine primitive-like bESCs are successfully obtained through single gene overexpression, so that the technical vacancy of cell induction in the field is filled, and the established cell line provides cell resources and reference for livestock stem cell research.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a primitive-like bovine embryonic stem cell system and an induction method and application thereof. Background Art

[0002] As one of the important livestock, cattle protein products not only provide rich nutrition for human diet, but also play a key supporting role in the global food industry chain. Among the various types of stem cell research and applications, embryonic stem cells are undoubtedly the most eye-catching. Naive embryonic stem cells, which have unlimited self-renewal ability and can differentiate into all cell types of adults, can be used for cattle gene editing optimization and embryo engineering breeding, which will greatly shorten the breeding cycle, speed up the pace of improving cattle breed traits, and have strong controllability and repeatability. However, the vast majority of bovine embryonic stem cells reported in the field are in an excited state, and there is an urgent need to develop new technologies for the transformation of cattle excited cells to the primitive state. Summary of the invention

[0003] The purpose of the present invention is to provide a primitive-like bovine embryonic stem cell line and its induction method and application, by overexpressing the JUP gene, activating the primitive signal regulation pathway inside bESCs, inducing bESCs to transform from an excited state to a primitive state, thereby exploring the physiological and biochemical characteristics of primitive bESCs.

[0004] The present invention provides a quasi-naive bovine embryonic stem cell line, wherein the bovine embryonic stem cell line overexpresses a gene JUP.

[0005] As a preferred embodiment, the number of the gene JUP is NM_001004024.3.

[0006] The present invention provides a method for inducing the above-mentioned primitive-like bovine embryonic stem cell line, comprising the following steps: inducing the bovine pluripotent stem cell line to overexpress the gene JUP to obtain the primitive-like bovine embryonic stem cell line.

[0007] As a preferred embodiment, the induction comprises the following steps: digesting the bovine pluripotent stem cell line into single cells, transfecting the JUP overexpression plasmid into the single cells, adding blasticidin for screening, and obtaining quasi-naive bovine embryonic stem cells that overexpress the JUP gene.

[0008] As a preferred embodiment, the dosage of the blasticidin is 3 to 15 μg / mL based on 1 mL of culture medium.

[0009] As a preferred embodiment, the screening includes screening 2 to 5 generations.

[0010] As a preferred embodiment, during the screening, each generation is screened for 2 to 5 days.

[0011] As a preferred embodiment, the induction temperature is 35-40°C.

[0012] As a preferred solution, the induced CO2 volume concentration is 3% to 10%.

[0013] The present invention provides the use of the above-mentioned quasi-naive bovine embryonic stem cell line or the quasi-naive bovine embryonic stem cell line induced by the above-mentioned induction method in cattle breeding.

[0014] Beneficial effects: The present invention provides a primitive-like bovine embryonic stem cell line, which overexpresses the gene JUP. The present invention obtains a primitive-like pluripotent stem cell line (bESCs) by overexpressing the gene JUP, screens and obtains a part of important primitive marker genes, and successfully obtains bovine primitive bESCs by overexpressing a single gene, which will fill the technical gap in the induction of such cells in the field, and the established cell line will provide cell resources and references for livestock stem cell research. The specific markers and related signal pathways of the primitive-like bESCs discovered by the present invention are effective references and guidelines for obtaining truly primitive bESCs in vitro. The gene JUP described in the present invention is an exogenous primitive control gene that overexpresses the physiological and biochemical changes that occur in bESCs during this process, and the effects of the gene JUP on specific signal pathways and markers, which is conducive to promoting the establishment of truly primitive bovine embryonic stem cells.

[0015] The present invention provides a method for inducing the above-mentioned primitive-like bovine embryonic stem cell line, comprising the following steps: inducing a bovine pluripotent stem cell line to overexpress the gene JUP to obtain the primitive-like bovine embryonic stem cell line. Currently, most studies focus on guiding the in vitro construction of primitive-like bESCs by changing environmental factors or exploring bovine early embryo-specific labels. The present invention, for the first time, induces bESCs to be converted into primitive-like bESCs by overexpressing the JUP gene alone. Compared with the prior art, the time consumption is greatly reduced, the method is simple, and the obtained cells can stably maintain pluripotency and self-renewal for a long time in vitro, which is of great significance for enriching the types of bovine embryonic stem cells and accelerating the application of stem cells in breeding.

[0016] The present invention provides the use of the above-mentioned quasi-naive bovine embryonic stem cell line or the quasi-naive bovine embryonic stem cell line induced by the above-mentioned induction method in cattle breeding. The quasi-naive bESCs obtained by the present invention tend to the original state in morphology and RNA level, and the cell state obtained by the present invention can be identified. The present invention can directly induce bESCs into quasi-naive bESCs in combination with gene editing technology, and the operation is simple and fast, which is of great significance for enriching the types of bovine embryonic stem cells and accelerating the application of stem cells in breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A flow chart for the induction of naive bovine embryonic stem cells derived from bovine pluripotent stem cells;

[0018] Figure 2 Flowchart for the construction of JUP gene overexpression plasmid;

[0019] Figure 3 This is the sequencing result of the JUP gene overexpression plasmid;

[0020] Figure 4 The naive bovine embryonic stem cells established in Example 1;

[0021] Figure 5 This is a diagram showing the results of WB verification of bovine embryonic stem cell lines and quasi-naive bovine embryonic stem cell lines using JUP protein antibodies and internal reference protein β-actin antibodies;

[0022] Figure 6 The results of using qPCR to detect the pluripotency and naive marker genes of bESCs and naive-like bESCs;

[0023] Figure 7 The volcano plot of differentially expressed genes in bESCs and naive-like bESCs with JUP gene overexpression in Example 1;

[0024] Figure 8 This is a KEGG pathway enrichment analysis diagram of differentially expressed genes in bESCs and naive-like bESCs in the present invention;

[0025] Fig. 9 This is a diagram showing the expression of core genes in the primitive and excited state characteristic signaling pathways of the bESCs and quasi-primitive bESCs transcriptomes in Example 1. DETAILED DESCRIPTION

[0026] The present invention provides a primitive bovine embryonic stem cell line, which overexpresses the gene JUP. As a preferred embodiment, the gene JUP is numbered NM_001004024.3. In the present invention, the gene JUP is transfected into bovine embryonic stem cells bESCs, which are used as a model to study the pluripotency regulation mechanism and cell characteristics of the primitive bESCs line.

[0027] The present invention provides a method for inducing the above-mentioned quasi-naive bovine embryonic stem cell line, comprising the following steps: inducing the bovine pluripotent stem cell line to overexpress the gene JUP to obtain the quasi-naive bovine embryonic stem cell line. The present invention constructs a bovine pluripotent embryonic stem cell line in a quasi-naive state by overexpressing the naive state control gene JUP in the bESCs cell line, thereby developing a bovine quasi-naive state ( -like) technology for establishing pluripotent stem cell lines.

[0028] As a preferred embodiment, a bovine pluripotent stem cell line (bESCs) established in the laboratory was used as the experimental material, and the primitive control gene JUP (NM_001004024.3) was overexpressed to obtain a primitive bovine embryonic stem cell line ( -likebESCs). As a specific embodiment, after culturing the bovine pluripotent stem cell line, the clones are digested with Tryple enzyme, and the clones are evenly blown with a gun tip to obtain single cells. The Tryple enzyme used in the present invention causes minimal damage to cells and can protect bovine embryonic stem cells to the greatest extent during the digestion process.

[0029] As a preferred embodiment, the gene JUP overexpression plasmid is transfected into the single cell, and blasticidin (BSD) is added for screening to obtain primitive bovine embryonic stem cells that overexpress the JUP gene. The transfection of the present invention may include electroporation. In the present invention, the dosage of the blasticidin can be 3 to 15 μg / mL, for example, 5 μg / mL or 10 μg / mL, based on 1 mL of culture medium. The blasticidin BSD resistance gene exists on the original plasmid of the gene JUP overexpression plasmid of the present invention. The plasmid is electroporated and inserted into the cell. The cell will have both the JUP gene overexpression fragment and the BSD resistance fragment, so the JUP overexpressing cell will have BSD resistance. When using exogenous BSD drug screening, only bESCs that are successfully electroporated will survive, and bESCs that are not successfully electroporated will be killed due to the action of the drug, so it is necessary to use blasticidin to purify the cell line.

[0030] During the screening described in the present invention, 2 to 5 generations, for example, 3 generations, can be screened; each generation can be screened for 2 to 5 days, for example, 3 days. Cells are generally subcultured every three days. After the drug is doubled, although JUP overexpressing bESCs have BSD resistance, the drug dosage is too large and the cell growth is affected, and it will gradually apoptosis over time, so three days of screening are selected as a node. In one embodiment, blasticidin (BSD) is added 48 hours after electroporation for drug screening, such as adding 5μg / mL blasticidin (BSD) for drug screening 48 hours after electroporation, and BSD is subcultured once 12 hours after addition. After subculture, the drug dosage is doubled, that is, 10μg / mL BSD is screened for three consecutive days. During this process, a large number of cells die, so it can only be screened for three days. In addition, attention should be paid to supplementing the trophoblast cells (Feeder); subculture again after three days, restore the normal BSD dosage, that is, 5μg / mL, and screen for another three days; after the drug screening, purified, primitive-like bovine embryonic stem cells overexpressing the JUP gene can be obtained.

[0031] As a preferred embodiment, during the whole induction process, the induction temperature is 35-40° C., for example, 38.5° C.; the induction CO2 volume concentration is 3%-10%, for example, 5%. In a specific embodiment, the mutagenesis culture can be carried out in an incubator.

[0032] To further illustrate the present invention, a quasi-naive bovine embryonic stem cell line and its induction method and application provided by the present invention are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] The bovine pluripotent stem cell line (bESCs) used in the present invention (public document: ZHAO L, GAO X, ZHENG Y, et al. Establishment of bovine expanded potential stem cells[J]. Proceedings of the National Academy of Sciences of the United States of America, 2021, 118(15):) was established in this laboratory in 2021 and is currently preserved in Laboratory 432, School of Life Sciences, Inner Mongolia University.

[0035] bESCs are transformed into naive bovine embryonic stem cells ( -likebESCs) induction method and process (induction process see Figure 1 ):

[0036] 1. Preparation of feeder cells (BFF-feeder):

[0037] Aliquot and preheat 2 mL of R stop solution (the composition of R stop solution is shown in Table 1) at 37°C in advance, spread 10 mL of gelatin in a 15 cm culture dish, add and shake well, and place in an incubator for 30 min; thaw bovine fetal fibroblast (BFF) cells in a 37°C water bath, transfer to the preheated R stop solution after the ice crystals melt, centrifuge at 1300 rpm for 3 min, and discard the supernatant; resuspend 20 mL of M10 culture solution (the composition of M10 culture solution is shown in Table 2) and inoculate into the pre-laid 15 cm culture dish, transfer to an incubator and culture for two to three days; transfer to 5 pre-laid 15 cm culture dishes after three days, and after two to three days, digest again and transfer to 20 15 cm culture dishes, and wait for two to three days again; when BFF grows to 90%, dilute mitomycin (MMC), add it to the dish for culturing BFF, and shake well. Treatment with mitomycin can stop BFF cells from dividing, leaving only the ability to provide a stable culture environment for stem cells. Therefore, feeder cells (BFF-feeder) can be obtained after treatment with MMC. Add MMC to a final concentration of 10 μg / mL and treat for at least 4 hours; after 4 hours, discard the culture medium, add 5 mL of PBS for washing, and then add 10 mL of TrypLE enzyme (TrypLE Select (1×), Gibco, 12563-011) for digestion for 6 minutes; after the end, add 20 mL of R stop solution to stop digestion, collect into a 50 mL centrifuge tube, and centrifuge at 1300 rpm for 3 minutes; after the end, discard the supernatant, collect the precipitate, resuspend with an appropriate amount of serum replacement (KSR), count the cells, and then use feeder cell freezing solution (the components of 1 mL feeder cell freezing solution are shown in Table 3) at a rate of 4.2×10 per cryopreservation tube. 6 Cryopreservation of BFF-feeder cells was performed on each cell. The next day, gel was plated for half an hour, and then a tube of BFF-feeder was thawed for observation. If there was no abnormal cell death or rapid proliferation rate on the second day of BFF-feeder inoculation, it means that BFF-feeder was successfully produced and subsequent experiments can be carried out.

[0038] Table 1 R stop solution composition

[0039] Components volume DMEM / F12 (Gibco, 11320033) 500mL KSR(Gibco,10828028) 57mL PS(Gibco,15140) 5.7mL

[0040] Table 2M10 culture medium composition

[0041] Components volume Knockoμt DMEM (Gibco, 10829018) 500mL FBS (VivaCell, 2210066) 57mL MEMNEAA (Gibco, 11140050) 5.7mL GlutaMAXSupplement(Gibco,35050061) 5.7mL PS(Gibco,15140) 5.7mL

[0042] Table 3 1mL feeder cell cryopreservation solution components

[0043] Components volume DMSO (Sigma, D2650) 1mL KSR(Gibco,10828028) 9mL

[0044] 2. Medium replacement and passaging of bovine pluripotent stem cells (bESCs):

[0045] Normal bESCs culture needs to be changed every day. Each time the medium is changed, the original culture medium is discarded first, and an appropriate amount of PBS buffer is added to shake and wash. After the end, the PBS is discarded and the bEPSCM culture medium is added (the composition of the bovine embryonic stem cell (bEPSCM) culture medium is shown in Tables 4 and 5). Generally, the volume of the 24-well single well to change the medium every day is 750μL, which depends on the cell proliferation state. Generally, the culture is subcultured once every three to four days. When subcultured, the original culture medium is still discarded first, PBS is added to wash and discard, and then the preheated Tryple enzyme is added. Generally, 300μL is added to a single well of 24 wells. After adding, the plate is transferred to the incubator for 4min, and then observed under the microscope. If it is observed that the cells are not suspended in a large area, continue to transfer to the incubator for digestion; but if 80% of the cells are observed to be suspended, 700μL of R stop solution can be added to terminate the digestion. After washing and mixing, transfer the suspension to a 1.5 mL centrifuge tube, centrifuge at 1300 rpm for 3 min, discard the supernatant, add 1 mL of bEPSCM culture medium to resuspend the precipitate, discard the pre-laid BFF-feeder, and after washing, the cells resuspended in 1 mL of bEPSCM culture medium can be inoculated on the BFF-feeder, shaken, and cultured. After three days of cell proliferation culture, 500 μL of bovine embryonic stem cell cryopreservation solution (1 mL of feeder layer cell cryopreservation solution composition table 6) can be used to freeze the unused bovine embryonic stem cells for subsequent use.

[0046] Table 4 Composition of bovine embryonic stem cell culture medium (bEPSCM)

[0047] Components <![CDATA[mTeSR TM 1(STEMCELL,85850)]]> 50mL CHIR99021 (Selleck, S2924) 30mM WH-4-023 (Selleck, S7565) 10mM IWR-1 (Selleck, S7086) 50mM Vitamin C (Sigma, 49752) 50 μg / mL ActivinA (R&D, 338-AC) 50 μg / mL SB590885 (Selleck, S2220) 10mM hLIF (Millipore, LIF1010) 5mM

[0048] Table 5 Bovine embryonic stem cell basal culture medium, mTeSR*1 culture medium composition

[0049] Components volume Basal Mediμm (STEMCELL, 85851) 400mL 5x Sμpplement(STEMCELL,85852) 100mL PS (Gibco, 15140) 5mL Mercaptoethanol (Gibco, 21985-023) 1mL

[0050] Table 6 1mL feeder cell cryopreservation solution components

[0051] Components volume DMSO (Sigma, D2650) 1mL KSR (Gibco, 10828028) 9mL

[0052] 3. Construction of JUP gene overexpression plasmid (see the plasmid construction process Figure 2 )

[0053] First, the total RNA of bESCs was extracted, and the total cDNA was obtained by inversion. The JUP fragment with MLU I and SAL I restriction sites was amplified using primers with restriction sites (primer information is shown in Table 7) (PCR-bJUP (MLUI-SALI)). Subsequently, the PB-CAG-K plasmid (nucleotide sequence is shown in SEQ ID NO.1) was double-digested with ECOR I and MLUI, and a 6239 base pair (bp) backbone fragment was recovered by agarose gel electrophoresis; the PB-CAG-V5-BSD-BFP-PA plasmid (nucleotide sequence is shown in SEQ ID NO.2) was double-digested with ECOR I and SAL I, and a 1819bp backbone fragment was recovered by agarose gel electrophoresis; the PCR-bJUP (MLUI-SALI) fragment was double-digested with MLU I and SAL I, and a 2275bp fragment was recovered. Finally, the above three were connected to obtain a complete JUP gene overexpression plasmid (JUP-V5-BSDR-BFP). This plasmid was then verified by DNA sequencing. The results are as follows Figure 3 .

[0054] from Figure 3 It can be seen that the JUP gene overexpression plasmid was correctly connected through sequencing by the company, and the blasticidin resistance gene (BSD) existed on the plasmid, which can be used as a screening marker to provide a basis for subsequent screening and purification.

[0055] Table 7 JUP gene amplification primers containing restriction sites

[0056]

[0057] 4. Induction of primitive bovine embryonic stem cells:

[0058] bESCs were seeded into bEPSCM basal culture medium (the components of bovine embryonic stem cell (bEPSCM) culture medium are shown in Tables 4 and 5) in a six-well plate, and the culture medium was discarded when the cells grew to 90% confluence. After washing with 1mL PBS, 1mL cell digestion solution (Accutase, ACC) was added along the wall and placed in the incubator for 6min. After the end, it was shaken for microscopic examination. After the embryonic stem cell clones were completely suspended, the supernatant was collected and centrifuged (1300rpm, 3min); after the centrifugation, the supernatant was discarded, 200μL of Tryple enzyme was added and mixed, and the cells were placed in the incubator for 4min. After the end, 400μLR stop solution was added to terminate the digestion and continued centrifugation (1300rpm, 3min); the purpose of the two-step digestion was to remove the feeder to prevent it from affecting the subsequent electroporation experiment; after centrifugation, the supernatant was discarded and the precipitate was resuspended with 100μL electroporation solution; 50μL electroporation solution was taken and 4.5μg JUP gene overexpression plasmid (JUP-V5-BSDR-BFP, which contains BSD resistance and blue fluorescence) and 1.5μg Pbase plasmid, after adding, put the above two volumes of electroporation solution together and mix them, add them into the electroporation cup along the wall for electroporation; after the electroporation is completed, use a pipette to suck out the electroporation solution, transfer it to a six-well plate containing bEPSCM culture medium and BFF-feeder, shake it well, and before the embryonic stem cells are connected to the BFF-feeder, discard the M10 liquid used to culture the BFF-feeder; change the medium normally after 4 hours, the purpose of changing the medium is to prevent the electroporation solution from causing cell death; the cells on the day of electroporation are recorded as the P0 generation, and normal bEPSCM culture medium (the composition of small factors in each 50mLmTeSR*1 during normal drug screening after JUP gene overexpression plasmid transfection is shown in Table 8) are cultured for two days, and after two days, culture medium containing 5μg / mL BSD is added for one day, and then passage is performed, and this passage is recorded as the P1 generation, and 5μg / mL BSD culture medium is used for culture; when reaching the P2 generation, the drug dosage is doubled, and culture medium containing 10μg / mL BSD is added (the composition of small factors in each 50mL mTeSR*1 during doubled drug screening after JUP gene overexpression plasmid transfection is shown in Table 9); after doubling the drug dosage, pay attention to observe the cell status. If large-scale clone death and a small number of cells occur, stop immediately and switch back to 5μg / mL BSD culture medium (the composition of small factors in each 50mL mTeSR*1 during normal drug screening after JUP gene overexpression plasmid transfection is shown in Table 8) for culture; if the cell number is large and there is no obvious death, continue to culture at 10μg / mL, but pay attention to supplementing BFF-feeder, generally every other day. At the P3 generation, bovine embryonic stem cells that overexpress the JUP gene can be seen in the original state. The cell status after the drug screening is completed is shown in Figure 4 .

[0059] from Figure 4 It can be clearly seen that the naive-like bESCs clone boundaries are clearer and the dome-shaped clone morphology is more obvious.

[0060] Table 8 Composition of small and medium factors per 50mL mTeSR*1 during normal drug screening after JUP gene overexpression plasmid transfection

[0061] Small factor composition concentration CHIR99021 (Selleck, S2924) 30mM WH-4-023 (Selleck, S7565) 10mM IWR-1 (Selleck, S7086) 50mM Vitamin C (Sigma, 49752) 50 μg / mL ActivinA (R&D, 338-AC) 50 μg / mL SB590885 (Selleck, S2220) 10mM hLIF (Millipore, LIF1010) 5mM BSD (Biyuntian, ST018) 5 μg / mL

[0062] Table 9 Composition of small and medium factors per 50mL mTeSR*1 during doubling of drug screening after JUP gene overexpression plasmid transfection

[0063] Small factor composition concentration CHIR99021 (Selleck, S2924) 30mM WH-4-023(Selleck,S7565) 10mM IWR-1(Selleck,S7086) 50mM Vitamin C (Sigma, 49752) 50 μg / mL ActivinA(R&D,338-AC) 50 μg / mL SB590885 (Selleck, S2220) 10mM hLIF (Millipore, LIF1010) 5mM BSD(Biyuntian,ST018) 10 μg / mL

[0064] Test Example 1

[0065] 1. Western Blot Analysis

[0066] First, extract the total protein. Collect two wells of the 6-well cell plate, add 200μL of cell protein lysis solution (Lysis), 2μL of protease inhibitor (PMSF), and 2μL of phosphatase inhibitor A / B to resuspend the cell pellet, mix thoroughly and lyse on ice for 25min, centrifuge at 16000rpm for 10min; take an appropriate amount of supernatant to measure the protein concentration by BCA method, add an appropriate amount of 6×LoadingBuffer to the remaining protein, boil at 100℃ for 5min, and store at -80℃ for standby; prepare concentrated gel and separation gel, put the solidified gel plate into the electrophoresis tank, pour in the electrophoresis buffer, apply the protein label (Marker) and the boiled protein sample, the protein loading amount is 30mg, after applying the sample, cover the lid, plug in the power, and start electrophoresis. The voltage and time of electrophoresis are 80V, 15min for concentrated gel; 150V, 9min for separation gel. After the electrophoresis is finished, take out the gel plate, carefully break the glass plate, separate the concentrated gel with a caliper, mark the front and back of the separation gel and soak it in the pre-prepared transfer solution for at least 30 minutes; prepare two sponges, soak them in the transfer solution with the gel for 30 minutes, cut another PVDF membrane of appropriate size, activate it in methanol for 15 seconds, wash it with ultrapure water for 10 minutes, and then transfer it to the transfer solution and soak it for 20 minutes; after the end, place the sponge-PVDF membrane-separation gel-sponge in order, and transfer it to the transfer instrument. 25V, transfer for 1h; after transfer, put the PVDF membrane into the blocking solution, 37℃, and block for 1h; after completion, cut the target band according to the marker, put it into the primary antibody diluent and incubate it at 4℃ overnight; the next day, wash it 3 times with 1×TBST, 10min / time, and incubate it in the secondary antibody diluent at 4℃ for 2h; after the secondary antibody incubation, wash it 3 times with 1×TBST, 10min / time, and soak it in the ECL colorimetric solution for 1min before detecting the target protein band on the machine. The WB results are shown in Figure 5 .

[0067] from Figure 5It can be seen that the JUP protein is overexpressed in the quasi-naive bovine embryonic stem cell line. The appearance of two bands is because the overexpressed JUP protein is connected to the V5 tag protein, causing the protein to become larger.

[0068] 2. Marker gene and protein detection:

[0069] After obtaining P3 primitive bovine embryonic stem cells, they were identified at the mRNA and protein levels. The experimental results are shown in Figure 6 .

[0070] from Figure 6 It can be seen that compared with bESCs, the expression of the classic pluripotent gene NANOG is upregulated in the naive bESCs; the naive marker genes KLF2, SALL4, TUBB3, ESRRB and TFPC2L1 remain unchanged, but TBX3, ZFP42 and KLF4 are upregulated, and the overall trend is a transition to the naive state.

[0071] mRNA level detection method: First, collect the cells. When the cells grow to 80% to 90% of the well, remove the culture medium, wash with PBS and discard, add 300μL Triple Enzyme to digest for 4 minutes, add 700μL Stop Solution to stop, centrifuge (1300rpm, 3min); discard the supernatant, resuspend in 1mL PBS, centrifuge again (4℃14000rpm, 5min), discard the supernatant after completion, and store at -20℃ for later use.

[0072] Extract total RNA from sample cells: The RNAfast200 total RNA extraction kit from Fijet Bio was used to extract mRNA from cells in this experiment. The specific process is as follows: first, add 1100μL of RA1 to resuspend the cell pellet. After thorough resuspending, add 500μL of RA2, mix by inverting and let stand for 1min; after completion, transfer to the adsorption column and centrifuge at 14000rpm for 1min; after completion, aspirate the liquid in the outer tube, add 500μL of washing solution, and centrifuge again (14000rpm, 1min); after completion, aspirate the liquid in the outer tube and repeat the previous step; after completion, place the adsorption column in a new centrifuge tube, add 30μL of elution solution in the center of the adsorption column membrane, and let it stand at room temperature for 1min; after completion, centrifuge (14000rpm, 1min) to obtain total RNA; detect RNA concentration on a UV spectrophotometer and mark it before proceeding to the next step of the experiment.

[0073] Reverse total RNA and obtain cDNA: The reagent used for mRNA reversal in this experiment is Novozymes' R223 mRNA reversal kit. The specific process is as follows: prepare the system in a PCR tube: 500ng RNA, 4μL 4×gDNA wiper Mix and an appropriate amount of water to make up to 16μL, mix well and place in a PCR instrument at 42℃ for 2min; after completion, add 4μL 5×HiScriptII qRT SuperMix II to the above mixture, mix again, and place in the PCR instrument at 50℃ for 15min and 85℃ for 5sec. After completion, obtain total mRNA and proceed to the next experiment.

[0074] Real-time fluorescence quantitative PCR (qPCR): The qPCR reagent used in this experiment is the Q711 qPCR kit of Novozymes. The specific process is as follows: prepare the system in the PCR tube: 2×ChamQ Universal SYBR qPCR MasterMix 10μL, upstream and downstream primers (primer information see: ZHAO L, GAO X, ZHENGY, et al. Establishment of bovine expanded potential stem cells [J]. Proceedings of the National Academy of Sciences of the United States of America, 2021, 118 (15):) 0.5μL each, cDNA 2μL, enzyme-free water 7μL, mix well; set the following cycle in the qPCR instrument: 95℃ pre-denaturation for 2min; 95℃ high temperature denaturation for 30sec, 58℃ low temperature annealing for 45sec, 72℃ extension for 30sec, a total of 30 cycles and then store at 4℃; convert the relative amount of gene expression according to the obtained Cq value.

[0075] 3. Transcriptome data analysis:

[0076] 10 bESCs and JUP-bESCs (primitive state) in good condition were collected. 7 Each cell was divided into three replicates for transcriptome library construction and NGS sequencing. After obtaining the data, fastp was used to filter out low-quality data in rawreads to obtain cleanreads; the cleandata obtained was compared with the bovine reference genome, and the front and back data were corrected; the gene expression was displayed in reads count and TPM format.

[0077] Differential gene analysis: Two groups, bESCs and quasi-naive bESCs, were set up and analyzed using the corrected and standardized TPM values. DESeq2 was used to analyze differential gene expression between bESCs and quasi-naive bESCs, and the threshold for differential genes was set to pvalue<0.05 and |log2 Foldchange|>1. Genes that met the threshold were identified as having significant differences and were represented in the form of volcano plots to visualize the differential genes. The results are shown in the figure. Figure 7 .

[0078] from Figure 7 It can be seen that the JUP gene is upregulated and successfully overexpressed, the primitive pluripotency marker genes KLF4, TBX3 and NANOG are upregulated; the DNA methylation-related gene DNMT3A is downregulated; primed marker genes such as SOX2 and OTX2 are downregulated. After the JUP gene is overexpressed, the cells tend to the primitive state.

[0079] KEGG analysis: KEGG enrichment analysis was performed on the significantly differentially expressed genes screened to explore the functions associated with the differentially expressed genes. KEGG signaling pathway enrichment analysis was performed using the gene annotation tool Metascape. The enrichment results were visualized using the ggplot2 package in R software. Figure 8 .

[0080] from Figure 8 It can be seen that the signaling pathways of bESCs and quasi-naive bESCs are differentially enriched in (Ko04550) stem cell pluripotency signaling regulation, (Ko04110) cell cycle, and (Ko04310) Wnt signaling pathways. These enrichment results are all related to the naive state, indicating that bESCs with overexpression of the JUP gene are quasi-naive bovine embryonic stem cell lines.

[0081] Heatmap analysis: First, the core genes regulating the JAK-STAT pathway and the IL6 signaling pathway were obtained in the GSEA database, and then queried in the obtained differential gene set. Then, the heatmap was drawn using the pheatmap in the R software. The results are shown in Fig. 9 .

[0082] from Fig. 9 It can be seen that the key genes of the JAK-STAT signaling pathway, a characteristic signaling pathway of the primitive state, and the IL6-mediated signaling pathway are enriched in the primitive-like bovine embryonic stem cells with overexpression of the JUP gene, while the key genes of the glycolysis characteristic signaling pathway of the excited state are enriched in the non-overexpression group. This shows that after the JUP gene is overexpressed, the cell state changes to the primitive state and is enriched with the signaling pathway characteristics related to the primitive state.

[0083] Comparative Example 1

[0084] NHSM and 5iLA culture media were used to establish naive bovine embryonic stem cells (see: Brinkhof B, van Tol HT, Groot Koerkamp MJ, Wubbolts RW, Haagsman HP, Roelen BA. Characterization of bovine embryos cultured under conditions appropriate for sustaining human pluripotency.PLoS One.2017Feb 27;12(2):e0172920.) The experimental results showed that the expression levels of ICM core pluripotency genes SOX2, OCT4 and NANOG in the obtained cells were downregulated compared with the control, and there was no change in the primitive marker genes DPPA2, TFCP2L1 and ESRRB, and the cells in the 5iLA culture system could not even be propagated for more than 5 generations. This shows that the primitive stem cell culture conditions for humans or mice cannot be directly used for the establishment of bovine primitive bESCs lines. The main reason for this result is that there is still no clear result on the regulation of specific signaling pathways and the identification of pluripotency markers for the establishment of primitive bESCs lines.

[0085] All the results of the present invention have three biological replicates, and relevant multiple tests have been carried out. The experimental results show that overexpression of the JUP gene and multiple rounds of drug screening can successfully obtain primitive-like bESCs with a single state. Compared with the methods of deriving the establishment of primitive-like bESCs by small factor combinations or bovine early embryo identification, the present invention is time-saving and effective, and can intuitively see the morphological changes and biological characteristics of cells before and after induction. In addition, the transcriptome data in the present invention will provide a theoretical basis for studying the role of different small factor combinations in inducing primitive stem cells.

[0086] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A quasi-naive bovine embryonic stem cell line, characterized in that: The bovine embryonic stem cell line overexpresses the gene JUP.

2. The bovine embryonic stem cell line according to claim 1, characterized in that The number of the gene JUP is NM_001004024.

3.

3. The method for inducing a naive bovine embryonic stem cell line according to claim 1 or 2, characterized in that: The method comprises the following steps: inducing the bovine pluripotent stem cell line to over-express the gene JUP to obtain the quasi-primitive bovine embryonic stem cell line.

4. The induction method according to claim 3, characterized in that: The induction comprises the following steps: digesting the bovine pluripotent stem cell line into single cells, transfecting the JUP overexpression plasmid into the single cells, adding blasticidin for screening, and obtaining primitive-like bovine embryonic stem cells overexpressing the JUP gene.

5. The induction method according to claim 3, characterized in that: Based on 1 mL of culture medium, the dosage of the blasticidin is 3 to 15 μg / mL.

6. The induction method according to claim 3, characterized in that: The screening includes screening 2 to 5 generations.

7. The induction method according to claim 6, characterized in that: During the screening, each generation is screened for 2 to 5 days.

8. The induction method according to claim 3, characterized in that: The induction temperature is 35-40°C.

9. The induction method according to claim 3, characterized in that: The induced CO2 volume concentration is 3% to 10%.

10. Use of the quasi-naive bovine embryonic stem cell line according to claim 1 or 2 or the quasi-naive bovine embryonic stem cell line induced by the induction method according to any one of claims 3 to 9 in cattle breeding.

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