Culture system and culture method for improving pluripotency-related gene expression of sheep embryonic stem cells

Abstract

This invention belongs to the field of cell biology technology, specifically relating to a culture system and method for enhancing the expression of genes related to pluripotency in sheep embryonic stem cells. The culture system uses mouse fetal fibroblasts as feeder cells and mTeSR... TM Plus was used as the basal culture medium, with 2.5 μM IWR-1 added as the ESC culture medium; the genes included were OCT4, SOX2, NANOG, DPPA3, LIN28, and SALL4. The culture method included: feeder cell preparation; isolation of sheep embryonic stem cells; and passage culture of sheep embryonic stem cells. The sheep embryonic stem cells obtained by this method expressed pluripotency-related genes at the transcriptomic and protein levels and had the ability to differentiate into all three germ layers. They can be widely used for the identification and screening of important genes during sheep embryonic development, the preparation of gene-edited animals, human disease research, and can also provide basic materials for sheep stem cell breeding.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to a culture system and method for enhancing the expression of genes related to pluripotency in sheep embryonic stem cells. Background Technology

[0002] Embryonic stem cells (ESCs, abbreviated as ES, EK, or ESC cells) are a type of cell isolated from early embryos (before gastrulation) or primitive gonads. They possess the characteristics of unlimited proliferation, self-renewal, and multi-lineage differentiation in vitro. Whether in vivo or in vitro, ESC cells can be induced to differentiate into almost all types of cells in the body.

[0003] Currently, ESCs have been extensively studied in mice and humans, and researchers have achieved significant results in using ESCs for functional studies of related genes and disease treatment. In goats and sheep, however, research progress on ESCs has been very limited due to the difficulty in establishing and cultivating them. Nevertheless, the cultivation of ESCs in large livestock is of great significance for gene function identification, screening, preparation of gene-edited animals, and stem cell breeding research. 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methylene-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (IWR-1) is an antagonist that inhibits the Wnt / β-catenin signaling pathway's end-anchored polymerase. Adding the small molecule IWR-1 to the culture medium inhibits β-catenin from entering the nucleus and interacting with TCF1 (T cytokine 1), thereby inhibiting GATA6 expression, suppressing ESC differentiation, and thus maintaining ESC self-renewal. Studies have shown that IWR-1 is essential for isolating and maintaining the stable growth of bovine and sheep ESCs. In the absence of IWR-1 in the culture medium, cells begin to differentiate and cease expressing the pluripotency-related genes OCT4 and SOX2. These studies provide insights for our in vitro isolation and culture of sheep embryonic stem cells.

[0004] Therefore, this invention provides a sheep embryonic stem cell culture system to provide a reference for the establishment of sheep and goat ESCs and the acquisition of culture conditions. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a culture system and method for enhancing the expression of genes related to pluripotency in sheep embryonic stem cells.

[0006] In a first aspect, the present invention provides a culture system for enhancing the expression of genes related to pluripotency in sheep embryonic stem cells, wherein the culture system uses mouse fetal fibroblasts as feeder cells and mTeSR... TMPlus (Stem Cell Technology, 100-0276) was used as the basic culture medium, and 2.5 μM IWR-1 (Sigma, I0161) was added as the ESC culture medium.

[0007] The genes include OCT4, SOX2, NANOG, DPPA3, LIN28, and SALL4.

[0008] Secondly, the present invention provides a method for culturing sheep embryonic stem cells using the system, wherein the sheep embryonic stem cells are co-cultured with the culture system.

[0009] Furthermore, the method for culturing sheep embryonic stem cells specifically includes the following steps:

[0010] Preparation of feeder layer cells;

[0011] Isolation of sheep embryonic stem cells;

[0012] Passaging of sheep embryonic stem cells.

[0013] Furthermore, in step (A1), the preparation of feeder cells includes the following steps:

[0014] When mouse fetal fibroblasts reached a confluence of over 75%, they were passaged at a ratio of 1:5.

[0015] After the cells have grown to confluence again after passage, the culture medium is replaced with a medium containing 10 μg / mL mitomycin C and treated for 3 h.

[0016] Discard the culture medium containing mitomycin C, wash the cells, and digest them with 0.25% trypsin to obtain a cell suspension;

[0017] The obtained cell suspension was diluted;

[0018] The diluted cell suspension was subjected to sequential treatments of 4℃ for 20 min, -20℃ for 40 min, and -80℃ for 24 h before being transferred to liquid nitrogen for storage.

[0019] Furthermore, the method used for isolating sheep embryonic stem cells is the whole embryo inoculation method, which specifically includes the following steps:

[0020] Sheep blastocysts with good morphology and obvious inner cell mass were selected for in vitro separation by ESCs, and the zona pellucida of the blastocysts was removed using acidic T1788-100mL solution.

[0021] Furthermore, the specific steps involved in propagation are as follows:

[0022] The sheep embryonic stem cell clones were removed from the original culture medium and washed once with D-PBS.

[0023] Discard D-PBS, digest cells with TrypLE Select, incubate at 37°C for 3 min, and terminate digestion with ESCs culture medium supplemented with Y-27632;

[0024] Change the ESC culture medium to one without Y-27632 every day from the second day onwards, and passage every 3-4 days.

[0025] Thirdly, the present invention provides a method for constructing an animal model of teratoma, wherein sheep embryonic stem cells are cultured using the method for culturing sheep embryonic stem cells, and the obtained sheep embryonic stem cells are injected subcutaneously into mice, and teratomas are formed after 10 weeks.

[0026] Fourthly, the present invention provides the application of the animal model constructed by the method for constructing a teratoma animal model in screening drugs for the treatment of teratoma.

[0027] The present invention has the following beneficial effects:

[0028] (1) Sheep embryonic stem cells cultured using the culture system and culture method of the present invention can form dome-shaped cell clones with clear edges. After multiple passages, their morphology does not change significantly. Pluripotency-related genes are stably expressed, the karyotype is normal, and alkaline phosphatase staining is positive. They have the ability to differentiate into three germ layers.

[0029] (2) The mTeSR Plus culture medium and IWR-1 involved in this invention are both commercial products that are easy to obtain. The culture medium preparation process is simple and convenient, and they are suitable for widespread use. Attached Figure Description

[0030] Figure 1 Determine the conditions and procedures for in vitro establishment of sheep embryonic stem cell lines.

[0031] Figure 2 Sheep ESCs isolated under different culture conditions (scale bar: 1000 μm).

[0032] Figure 3 Morphology of sheep ESCs under different culture conditions after 10 passages (scale bar: 1000 μm).

[0033] Figure 4 : Sheep ESCs isolated from different feeder layers (scale bar: 1000 μm).

[0034] Figure 5Morphology (scale bar: 1000 μm) and alkaline phosphatase (AP) staining (scale bar: 200 μm) of TePR-sESCs cells at different generations. a is TePR-sESCs cells of generation 10, and b is TePR-sESCs cells of generation 47.

[0035] Figure 6 Cell cycle detection results and quantitative statistics of TePR-sESCs (A) and sheep fibroblasts (B) (C). * indicates significant difference, P<0.05; ** indicates extremely significant difference, P<0.01; ns indicates no significant difference, P>0.05. Each experiment was repeated three times.

[0036] Figure 7 Images showing the expression levels of pluripotency genes in SFF, TePR-sESCs (P10), and TePR-sESCs (P50) cells. * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01); ns indicates no significant difference (P>0.05). Each experiment was repeated three times.

[0037] Figure 8 Images of TePR-sESCs (P10) and TePR-sESCs (P47), and OCT4, SOX2, and NANOG cell immunofluorescence staining (scale bar: 200 μm).

[0038] Figure 9 Images of TePR-sESCs (P10) and TePR-sESCs (P47), and immunofluorescence staining of SSEA4, TRA-1-60, and TRA-1-81 cells (scale bar: 200 μm).

[0039] Figure 10 Images of TePR-sESCs karyotype (a) and embryoid (b).

[0040] Figure 11 Images showing mRNA gene expression in the three germ layers of the embryoid body. * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01); ns indicates no significant difference (P>0.05). Each experiment was repeated three times.

[0041] Figure 12 HE staining image of teratoma differentiation of the three germ layers: A: endoderm, B: mesoderm, C: ectoderm. Detailed Implementation

[0042] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0043] Example 1: Preparation of mouse fetal fibroblast feeder cells

[0044] 1. Preparation of feeder cells

[0045] 1.1 Isolation of mouse fetal fibroblasts

[0046] (1) Take the fetus of a pregnant female mouse that is 12 days pregnant, remove the fetus from the gestation sac, and then remove the head, limbs, internal organs and tail with tweezers and scissors. The remaining tissue is placed in the prepared DuPont phosphate buffer (D-PBS) for washing.

[0047] (2) Cut the cleaned tissue into small pieces with scissors, digest with 0.25% trypsin at 37°C for 10-15 min, blow the tissue pieces with a 15 mL pipette to disperse them, continue digestion for 5 min, and add mouse fetal fibroblast culture medium to stop digestion.

[0048] (3) Collect the cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, add an appropriate amount of culture medium to resuspend the cells, and seed them into a 100 mm culture dish;

[0049] (4) Frozen after the cells have grown to full size.

[0050] 1.2 Preparation of feeder layer cells

[0051] (1) Resuscitate frozen mouse fetal fibroblasts and inoculate them into 100 mm cell culture dishes. When the cell confluence reaches more than 75%, passage the cells at a ratio of 1:5.

[0052] (2) After the cells have grown to full confluence again after passage, the culture medium is replaced with a culture medium containing 10 μg / mL mitomycin C (Selleck, S8146) and treated for 3 h.

[0053] (3) Discard the culture medium containing mitomycin C, wash 5 times with preheated D-PBS, and digest with 0.25% trypsin in a 37°C, 5% CO2 incubator for 3 min to obtain cell suspension;

[0054] (4) Count the obtained cell suspension and dilute the cells to 4.8 × 10⁻⁶ cells per mL using cryopreservation solution. 5 Fibroblasts were aliquoted into cryovials with 900 μL of cell suspension and 100 μL of DMSO per tube for cryopreservation.

[0055] (5) After 20 min at 4℃, 40 min at -20℃, and 24 h at -80℃, transfer to liquid nitrogen for storage.

[0056] Example 2: Preparation of sheep fetal fibroblast feeder cells

[0057] The specific procedures for primary separation of sheep fetal fibroblasts are as follows:

[0058] (1) The 45-day fetus was placed in D-PBS containing 5× bispecific antibody and brought back to the laboratory. It was then rinsed three times with D-PBS containing 1× bispecific antibody and alcohol, respectively.

[0059] (2) Remove the fetus’s head, limbs and internal organs in a petri dish, leaving only the torso in the petri dish;

[0060] (3) Cut the remaining tissue into 1mm pieces. 3 Transfer the tissue blocks from the left and right sides to centrifuge tubes and wash them twice each with D-PBS and DMEM containing 1× anti-adhesive (washing is optional if conditions are insufficient, but aseptic operation must be observed to avoid contamination).

[0061] (4) Spread the tissue blocks evenly in the culture dish, dry them in the incubator for 0.5-1h, slowly add DMEM medium containing 10% FBS and 1× double antibiotics, and incubate at 37℃ and 5% CO2. After 48h, remove the tissue blocks that have not adhered to the wall and replace the medium.

[0062] (5) When the cell confluence reaches more than 80%, remove the tissue block and culture medium, wash twice with D-PBS, digest with 0.25% trypsin for about 2 minutes, add serum-containing culture medium to stop digestion, blow the cells off the bottom of the dish and collect them into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, resuspend the cells with fresh culture medium, and passage them at a ratio of 1:3 to 1:5.

[0063] The preparation of feeder cells is the same as that of feeder cells prepared using mouse fetal fibroblasts, as detailed in Example 1.

[0064] Example 3: Determination of in vitro establishment conditions for sheep embryonic stem cells

[0065] Three published ESC culture systems—NBFR (Soto et al. 2021), bEPSCM (Zhao et al. 2021), and 3i / LAF (Zhi et al. 2022)—and a TePR culture system (using mTeSR PLUS as the basal medium supplemented with the Wnt signaling pathway inhibitor IWR-1)—were used to establish cell lines. Figure 1 ,2).

[0066] This invention employs a whole-embryo inoculation method for establishing ESCs, and the specific procedures are as follows:

[0067] (1) Replace the feeder cell culture medium prepared one day in advance with ESCs culture medium, add 500 μL / well culture medium to the 4-well plate, and add Y-27632 to a final concentration of 10 μM.

[0068] (2) Place the blastocyst in an acidic liquid and, under a stereomicroscope, repeatedly blow the embryo with a pipette to remove the zona pellucida.

[0069] (3) Transfer the blastocysts with the zona pellucida removed to a dish containing preheated ESCs culture medium and wash 2-3 times;

[0070] (4) Transfer the blastocysts to a culture medium containing Y-27632 and incubate them at 37°C and 5% CO2 for 48 hours.

[0071] (5) On the third day, gently discard the culture medium with a pipette and add fresh culture medium containing Y-27632. Then change the ESCs culture medium containing Y-27632 every day until cell clones grow.

[0072] (6) Once the clones have grown to a certain size, discard the culture medium, add 200 μL TrypLE select, incubate at 37°C for 3 min, gently pipette to transfer the cell suspension to a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells in fresh ESCs culture medium, and seed them onto newly prepared feeder cells at a 1:1 ratio. On the day of seeding, add Y-27632 to a final concentration of 10 μM. On the second day, replace the culture medium with ESCs culture medium without Y-27632 (see the isolated sESCs). Figure 2 ).

[0073] The results showed that when mouse fetal fibroblasts were used as feeder cells, well-defined cell clones with morphology consistent with those reported in sheep and other species ESCs could be obtained under all four culture conditions. Figure 2 The obtained cells were named NBFR-sESCs, bEPSCM-sESCs, 3i / LAF-sESCs, and TePR-sESCs, respectively. Figure 2 However, after multiple cell passages (more than 10 generations), it was found that the proliferation of three cell lines, NBFR-sESCs, bEPSC-sESCs, and 3iLAF-sESCs, was slow, and the cells gradually died. Figure 3This indicates that there are still some differences in embryonic stem cell isolation and culture systems among different species. The culture conditions for bovine and porcine embryonic stem cells are not suitable for the isolation and culture of sheep embryonic stem cells. TePR-sESCs, however, can be stably passaged and grown. When sheep fetal fibroblasts are used as feeder cells, no obvious cell clones appear. Figure 4 ).

[0074] Example 4: Establishment of sheep embryonic stem cell lines

[0075] 1. In vitro isolation of sheep embryonic stem cells

[0076] Sheep blastocysts with good morphology and obvious inner cell mass were selected for in vitro ESC isolation. After removing the zona pellucida of the blastocysts using acidic T1788-100mL solution, they were placed on mouse fetal fibroblast feeder cells and established using the TePR culture system (based on mTeSR PLUS culture medium supplemented with the Wnt signaling pathway inhibitor IWR-1). The whole embryo seeding method was used for in vitro cell isolation and establishment. For details of the operation, please refer to Example 3.

[0077] 2. Passaging of sheep embryonic stem cells

[0078] (1) When the cell clones have grown to a certain size, discard the original culture medium with a pipette and wash once with D-PBS.

[0079] (2) Discard D-PBS, add 200 μL TrypLE Select to each well of a 4-well plate to digest cells, and incubate the culture plate at 37°C for 3 min after adding TrypLE Select.

[0080] (3) Gently pipette the cells to transfer the cell suspension to a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells in ESCs culture medium, pipette evenly to avoid large cell clumps, and seed the cells onto the prepared feeder cell layer. Add Y-27632 to a final concentration of 10 μM;

[0081] Change the culture medium to one without Y-27632 every day from the second day onwards. Generally, passage the cells every 3-4 days.

[0082] 3. Cryopreservation and thawing of sheep embryonic stem cells

[0083] (1) Sheep ESCs cryopreservation:

[0084] After digesting the cells, collect the cell suspension, centrifuge at 1000 rpm for 3 min, discard the supernatant, and resuspend the cells in 900 μL of sESC culture medium to DMSO at a ratio of 9:1. Transfer the cell suspension to cryovials, add 100 μL of DMSO, mix well by pipetting, label the cryovials, and place them in a gradient cryopreservation box at -80°C. After 24 h, transfer them to liquid nitrogen for long-term storage.

[0085] (2) ESCs recovery in sheep:

[0086] Preheat the water bath to 37°C. Remove the cells to be thawed from the liquid nitrogen tank and quickly place them in the water bath. Shake the cryovials until they are completely thawed. Transfer the liquid in the cryovials to a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add fresh sESCs culture medium, resuspend the cells, seed them onto the prepared feeder layer cells, and add Y-27632 to a final concentration of 10 μL. The next day, replace the medium with culture medium without Y-27632, and change the medium daily thereafter.

[0087] Example 5: Identification of sheep embryonic stem cells

[0088] 1. Morphological observation and alkaline phosphatase (AP) staining of sheep embryonic stem cells

[0089] First, the morphology of the obtained TePR-sESCs was observed. It was found that TePR-sESCs cells had small diameters, large nuclei, prominent nucleoli, little cytoplasm, and relatively large nucleoplasm, exhibiting a dome-shaped growth pattern. The cell clones had clear edges, a relatively smooth surface, and were refractive. The ESCs in the state have the same morphology, and their morphology has not changed significantly after multiple generations, see Figure 5 .

[0090] When the confluence of sheep embryonic stem cells reached 70%-80%, fix them with 4% paraformaldehyde (PFA) for 1 min, and strictly follow the instructions of the alkaline phosphatase staining kit (Beyotime, P0321S), incubating at room temperature in the dark for about 15 min, followed by microscopic examination. The results are shown below. Figure 3 AP staining results showed that TePR-sESCs were positive for alkaline phosphatase.

[0091] 2. Analysis of the proliferation capacity of TePR-sESCs

[0092] Sheep fetal fibroblasts (SFF) and TePR-sESCs were inoculated into 12-well plates, with three replicates of each. After culturing for 3 days, cells were collected and analyzed using a cell cycle assay kit, following the kit's instructions. Results are shown below. Figure 6The results showed that, compared with sheep fibroblasts, the number of TePR-sESCs cells in the G2 and S phases was significantly higher than that in SFF (P<0.01).

[0093] 3. Expression analysis of TePR-sESCs pluripotency marker genes

[0094] Total RNA was extracted from sheep embryonic stem cells P10 and P48, respectively, and hydrolyzed with RNase-free water. After measuring the concentration and purity of the RNA, cDNA libraries were synthesized via reverse transcription. The expression of the sheep embryonic stem cell pluripotency genes OCT4, SOX2, and NANOG was detected by qRT-PCR. Results are shown below. Figure 7 As shown, the mRNA expression levels of pluripotency genes OCT4, SOX2, and NANOG in sheep embryonic stem cells from low generation (P10) and high generation (P50) were significantly higher than those in SFF.

[0095] When the confluence of TePR-sESCs reached approximately 80%, the cells were fixed with 4% PFA for 10 min, and then immunofluorescence staining was performed to detect the expression of the pluripotent genes OCT4, SOX2, and NANOG. The results showed that OCT4, SOX2, and NANOG proteins were expressed normally (see...). Figure 8 Cell surface markers SSEA4, TRA-1-60, and TRA-1-81 are normally expressed (see [link to relevant documentation]). Figure 9 ).

[0096] 4. Analysis of chromosome number and in vitro differentiation capacity of TePR-sESCs

[0097] Karyotype analysis was performed on TePR-sESCs to identify whether chromosomal aberrations occurred. Results showed a normal chromosome number, 2n = 54 (see...). Figure 10 a) can be used for subsequent experiments.

[0098] ESCs were digested into single cells and seeded at a specific density in U-shaped culture dishes with low adsorption density. The cells were then cultured in suspension for 7 days, with the medium changed every other day. After 7 days, the resulting embryoid bodies were transferred to gelatin-coated cell culture plates and cultured for another 14-28 days. The results are as follows... Figure 11 As shown, the cells can form embryoid bodies with a spherical structure. After in vitro differentiation, qRT-PCR detection of the marker genes of the three germ layers revealed that TePR-sESCs have the potential to differentiate into the three germ layers in vitro. Figure 10 b, Figure 11 ).

[0099] 5. In vivo differentiated teratoma

[0100] To further verify the differentiation potential of TePR-sESCs, TePR-sESCs were injected subcutaneously into immunized mice. Ten weeks after injection, teratomas formed. The teratomas were removed and analyzed by immunohistochemistry. The results showed that the formed teratomas had a three-layer germ layer structure. Figure 12 ).

[0101] References:

[0102] (1)Chen G, Yin S, Zeng H, Li H, Wan X. Regulation of Embryonic Stem CellSelf-Renewal. Life. 2022; 12(8): 1151.

[0103] (2)Vilarino M,Alba Soto D,Soledad Bogliotti Y,Yu L,Zhang Y,Wang C,Paulson E,Zhong C,Jin M,Carlos Izpisua Belmonte J,Wu J,Juan Ross P.Derivation of sheep embryonic stem cells under optimized conditions.Reproduction.2020Nov;160(5):761-772.

[0104] (3)Soto DA, Navarro M, Zheng C, Halstead MM, Zhou C, Guiltinan C, Wu J, RossPJ. Simplification of culture conditions and feeder-free expansion of bovineembryonic stem cells. Sci Rep. 2021 May 26;11(1):11045.

[0105] (4)Zhao L, Gao X, Zheng Y, Wang Z, Zhao G, Ren J, Zhang J, Wu J, Wu B, Chen Y, Sun W, Li Y, Su J, Ding Y, Gao Y, Liu M, Bai X, Sun L, Cao G, Tang F, Bao S, Liu P, Li Apr 13;118(15):e2018505118.

[0106] (5) Zhi M, Zhang J, Tang Q, Yu D, Gao S, Gao D, Liu P, Guo J, Hai T, Gao J, Cao S, Zhao Z, Li C, Weng X, He M, Chen T, Wang Y, Long K, Jiao D, Li G, Zhang J, Liu Y, Lin Y, Pang D, Zhu Q, Chen N, Huang J, Chen X, Yao Y, Yang J, Xie Z, Huang X, Liu M, Zhang R, Li Q, Miao Y, Tian J, Huang X, Ouyang H, Liu B, Xie W, Zhou Q, Wei H, Liu Z, Zheng C, Li M, Han J. Generation and characterization of stable pig pregastrulation epiblast stem cell lines. Cell Res. 2022 Apr;32(4):383-400.

[0107] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0108] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for constructing an animal model of teratoma, characterized in that, include: Sheep embryonic stem cells with high expression of pluripotency-related genes were cultured, and the resulting sheep embryonic stem cells were injected subcutaneously into mice. After 10 weeks, teratomas formed. The genes include... OCT4, SOX2 , NANOG, DPPA3, LIN28 and SALL4 ; The specific steps for obtaining the sheep embryonic stem cells include: Preparation of mouse fetal fibroblast feeder cells; ESC culture medium preparation: with mTeSR TM Plus was used as the basic culture medium, and 2.5 µM IWR-1 was added as the ESCs culture medium. ESCs system establishment: (1) Replace the mouse fetal fibroblast feeder cell culture medium prepared one day in advance with ESCs culture medium, add 500 μL / well culture medium to the 4-well plate, and add Y-27632 to a final concentration of 10 μM. (2) Place the sheep blastocyst in acidic liquid and blow the embryo repeatedly with a pipette under a stereomicroscope to remove the zona pellucida. (3) Transfer the blastocysts with the zona pellucida removed to a dish containing preheated ESCs culture medium and wash 2-3 times; (4) The blastocysts were transferred to a culture medium containing Y-27632 and cultured in an incubator at 37°C and 5% CO2 for 48 h. (5) On the third day, gently discard the culture medium with a pipette and add fresh culture medium containing Y-27632. Then change the ESCs culture medium containing Y-27632 every day until cell clones grow. (6) After the clones have grown to a certain size, discard the culture medium, add 200 μL TrypLE select, incubate at 37℃ for 3 min, gently pipette and transfer the cell suspension to a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells in fresh ESCs culture medium, and inoculate them into newly prepared mouse fetal fibroblast feeder cells at a 1:1 ratio. On the day of inoculation, add Y-27632 to a final concentration of 10 μM. On the second day and thereafter, replace the ESCs culture medium without Y-27632 every day, and passage once every 3-4 days.

2. The method according to claim 1, characterized in that, The preparation of mouse fetal fibroblast feeder cells includes the following steps: When mouse fetal fibroblasts reached a confluence of over 75%, they were passaged at a ratio of 1:

5. After the cells have grown to confluence again after passage, the culture medium is replaced with a medium containing 10 µg / mL mitomycin C and treated for 3 hours. Discard the culture medium containing mitomycin C, wash the cells, and digest them with 0.25% trypsin to obtain a cell suspension; The obtained cell suspension was diluted; The diluted cell suspension was subjected to sequential treatments at 4℃ for 20 min, -20℃ for 40 min, and -80℃ for 24 h before being transferred to liquid nitrogen for storage.

3. The application of the animal model constructed by the method of claim 1 in screening drugs for the treatment of teratoma.

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