CN117417883A - Culture system and culture method for improving sheep embryo stem cell multipotency related gene expression - Google Patents

Abstract

The invention belongs to the technical field of cell biology, and particularly relates to a culture system and a culture method for improving sheep embryo stem cell multipotency related gene expression. The culture system takes mouse fetal fibroblasts as feeder cells and mTESR ^TM Plus is used as basic culture solution, and 2.5 mu M IWR-1 is added as ESCs culture solution; the genes include OCT4, SOX2, NANOG, DPPA3, LIN28, and SALL4. The culture method comprises the following steps: preparing feeder cells; separating sheep embryo stem cells; and (5) subculturing sheep embryo stem cells. The sheep embryo stem cells obtained by the culture method express the genes related to pluripotency at transcriptome and protein level and have functions of expressing the genes towards three germ layersThe differentiation capability can be widely applied to identification and screening of important genes in the sheep embryo development process, preparation of gene editing animals, research of human diseases, basic materials for sheep stem cell breeding and the like.

Description

Culture system and culture method for improving sheep embryo stem cell multipotency related gene expression

Technical Field

The invention belongs to the technical field of cell biology, and particularly relates to a culture system and a culture method for improving sheep embryo stem cell multipotency related gene expression.

Background

Embryonic stem cells (embryonic stem cell, ESCs, abbreviated as ES, EK or ESC) are a class of cells isolated from early embryo (pre-gastrula) or primordial gonads and have the properties of in vitro culture immortalization, self-renewal and multipotency. ESC cells can be induced to differentiate into almost all types of cells of the body, both in an in vivo and in vitro environment.

At present, ESCs have been studied in mice and humans in a large number, and researchers have achieved great success in functional studies and disease treatment of related genes by using ESCs. In goats and sheep, research into ESCs has been very limited due to the fact that the construction and culture conditions of ESCs are difficult to fuze. However, the cultivation of large livestock ESCs is of great significance for research in gene function identification, screening, gene editing animal preparation, and stem cell breeding. 4- (1, 3a,4,7 a-hexahydro-1, 3-dioxo-4, 7-methylene-2H-isoindol-2-yl) -N-8-quinolinyl-benzamide (IWR-1) is a terminal anchor polymerase antagonist that inhibits the Wnt/β -catenin signaling pathway, and the addition of 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, inhibiting ESCs differentiation, and thus maintaining self-renewal of ESCs. Studies have shown that IWR-1 is essential in isolating, maintaining stable growth of bovine and ovine ESCs, the lack of IWR-1 in the medium, the cells will begin to differentiate, and the pluripotency-related genes OCT4 and SOX2 are no longer expressed. The research provides ideas for in vitro separation and culture of sheep embryo stem cells.

Therefore, the invention provides a culture system of sheep embryo stem cells, which is used for providing references for constructing sheep and goat ESCs and obtaining culture conditions.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a culture system and a culture method for improving the expression of the sheep embryo stem cell multipotency related genes.

In a first aspect, the present invention provides a culture system for increasing expression of a gene associated with pluripotency of sheep embryonic stem cells, the culture system comprising mouse fetal fibroblasts as feeder cells and mTESR ^TM Plus (Stem Cell Technology, 100-0276) as the base broth and 2.5. Mu.M IWR-1 (Sigma, I0161) as the ESCs broth;

the genes include OCT4, SOX2, NANOG, DPPA3, LIN28, and SALL4.

In a second aspect, the present invention provides a method for culturing sheep embryo stem cells using the system, the sheep embryo stem cells being co-cultured with the culture system.

Further, the method for culturing sheep embryo stem cells specifically comprises the following steps:

preparing feeder cells;

separating sheep embryo stem cells;

and (5) subculturing sheep embryo stem cells.

Further, in step (A1), the feeder cells are prepared by the steps of:

when the mouse fetal fibroblast is cultured until the cell confluence reaches more than 75%, the method comprises the following steps of 1:5, carrying out cell passage according to the proportion;

after the cells grow fully again after passage, the culture solution is replaced by the culture solution containing 10 mug/mL mitomycin C, and the culture solution is treated for 3 hours;

discarding the culture solution containing mitomycin C, washing, and then digesting with 0.25% pancreatin to obtain a cell suspension;

diluting the obtained cell suspension;

the diluted cell suspension is sequentially treated at 4 ℃ for 20min, 20 ℃ for 40min and 80 ℃ for 24h and then transferred into liquid nitrogen for preservation.

Furthermore, the method for separating sheep embryo stem cells is a full embryo inoculation method, and specifically comprises the following steps:

sheep blasts with good morphology and obvious inner cell mass are selected for ESCs in vitro separation, and blastula zona pellucida is removed by using acidic desk top liquid (Sigma, T1788-100 mL).

Further, the subculture specifically comprises the following steps:

the sheep embryo stem cell clone removes the original culture solution, and the sheep embryo stem cell clone is washed once with D-PBS;

discarding the D-PBS, digesting the cells with TrypLE Select, incubating at 37 ℃ for 3min, and stopping digestion with ESCs culture medium added with Y-27632;

the ESCs culture without Y-27632 was changed the next day and thereafter, and passaged once for 3-4 days.

In a third aspect, the present invention provides a method for constructing a teratoma animal model, culturing sheep embryo stem cells by using the method for culturing sheep embryo stem cells, injecting the obtained sheep embryo stem cells into the subcutaneous of a mouse, and forming teratomas after 10 weeks.

In a fourth aspect, the invention provides application of an animal model constructed by the method for constructing a teratoma animal model in screening teratoma treatment medicines.

The invention has the following beneficial effects:

(1) The sheep embryo stem cells obtained by adopting the culture system and the culture method can form dome-shaped cell clones with clear edges, the morphology of the sheep embryo stem cells is not obviously changed after multiple passages, the pluripotency related genes are stably expressed, the karyotype is normal, and the alkaline phosphatase staining is positive, so that the sheep embryo stem cells have the capability of differentiating into three germ layers.

(2) The mTESR Plus culture medium and the IWR-1 are all commercial products, are easy to obtain, have simple and convenient preparation process of culture solution, and are suitable for popularization and use.

Drawings

Fig. 1: and determining the in vitro establishment condition flow of the sheep embryo stem cells.

Fig. 2: sheep ESCs were isolated under different culture conditions (scale: 1000 μm).

Fig. 3: sheep ESCs morphology (scale: 1000 μm) under different culture conditions after 10 passages of the cells.

Fig. 4: sheep ESCs were isolated from different feeder cells (scale: 1000 μm).

Fig. 5: different algebraic TePR-sESCs cell morphologies (ruler: 1000 μm) and Alkaline Phosphatase (AP) staining (ruler: 200 μm), a being 10 th generation TePR-sESCs cells, b being 47 th generation TePR-sESCs cells.

Fig. 6: cell cycle test results and quantitative statistics (C) of TePR-sESCs (A) and sheep fibroblasts (B). * Representing significant differences, P <0.05; * Representing the difference being very significant, P <0.01; ns stands for no significant difference, P >0.05. Each set of experiments was repeated three times.

Fig. 7: SFF, tePR-sESCs (P10) and TePR-sESCs (P50) cell multipotent gene expression level pictures. * Representing significant differences, P <0.05; * Representing the difference being very significant, P <0.01; ns stands for no significant difference, P >0.05. Each set of experiments was repeated three times.

Fig. 8: tePR-sESCs (P10) and TePR-sESCs (P47), OCT4, SOX2, NANOG cell immunofluorescence staining pictures (scale: 200 μm).

Fig. 9: tePR-sESCs (P10) and TePR-sESCs (P47), SSEA4, TRA-1-60, TRA-1-81 cell immunofluorescence staining pictures (scale: 200 μm).

Fig. 10: pictures of TePR-sESCs karyotype (a) and embryoid body (b).

Fig. 11: picture of embryoid body trigermal mRNA gene expression. * Representing significant differences, P <0.05; * Representing the difference being very significant, P <0.01; ns stands for no significant difference, P >0.05. Each set of experiments was repeated three times.

Fig. 12: teratoma tricodermic differentiation HE staining picture, a: endoderm, B: mesoderm, C: ectoderm.

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

Example 1 preparation of mouse fetal fibroblast feeder layer cells

1. Preparation of feeder cells

1.1 isolation of fetal mouse fibroblasts

(1) Taking pregnant 12D female mouse fetus, taking the fetal mouse out of embryo sac, removing head, limbs, viscera and tail with forceps and scissors, and cleaning the rest tissue in prepared Dunaliella phosphate buffer (D-PBS);

(2) Shearing the cleaned tissue with scissors, digesting with 0.25% pancreatin at 37deg.C for 10-15min, blowing tissue blocks with 15mL pipette to disperse, continuing digestion for 5min, and adding mouse fetal fibroblast culture solution to stop digestion;

(3) Collecting cell suspension, centrifuging at 1000rpm for 5min, discarding supernatant, adding appropriate amount of culture solution to resuspend cells, and inoculating into a 100mm culture dish;

(4) Freezing and preserving after the cells grow fully.

1.2 preparation of feeder cells

(1) Resuscitate the cryopreserved mouse fetal fibroblasts, inoculate in 100mm cell culture dish, when the cell confluency reaches above 75%, according to 1:5, carrying out cell passage according to the proportion;

(2) After the cells grow up again after passage, the culture solution is replaced by a culture solution containing 10 mug/mL mitomycin C (Selleck, S8146) and treated for 3 hours;

(3) Discarding culture solution containing mitomycin C, washing with preheated D-PBS for 5 times, and washing with 0.25% pancreatin at 37deg.C and 5% CO ₂ Digesting for 3min in an incubator to obtain a cell suspension;

(4) The cell suspension obtained was counted and the cells were diluted to 4.8X10 per 1mL with frozen stock ⁵ The fibroblast is divided into freezing tubes by 900 mu L of cell suspension and 100 mu L of DMSO per tube for cell freezing;

(5) Transferring to liquid nitrogen for preservation at-20deg.C for 20min and-20deg.C for 40min and-80deg.C for 24 hr.

Example 2: sheep fetal fibroblast feeder cell preparation

The specific operation of the primary separation of the sheep fetus by the fiber forming method is as follows:

(1) The 45D fetus is put into D-PBS containing 5 Xdiabody and brought back to a laboratory, and the fetus is washed three times with the D-PBS containing 1 Xdiabody and alcohol respectively;

(2) Removing the head, limbs and viscera of the fetus in the culture dish, and only keeping the trunk in the culture dish;

(3) Cutting the remaining tissue to 1mm ³ Transferring left and right tissue blocks into a centrifuge tube, and cleaning for 2 times by using 1 Xdouble-antibody-containing D-PBS and DMEM (when the conditions are insufficient, the cleaning can be omitted, but the aseptic operation must be taken care of, so that pollution is avoided);

(4) Spreading the tissue block in culture dish, drying in incubator for 0.5-1 hr, slowly adding DMEM medium containing 10% FBS and 1×double antibody, and 5% CO at 37deg.C ₂ Culturing under the condition, removing the tissue blocks which are not adhered to the surface after 48 hours, and replacing the culture medium;

(5) When the cell confluence reaches more than 80%, removing tissue blocks and a culture medium, washing twice by using D-PBS, digesting for about 2min by using 0.25% pancreatin, adding a culture medium containing serum to stop digestion, blowing off cells from a dish, collecting the cells to a centrifuge tube, centrifuging at 1000rpm for 5min, re-suspending the cells by using a fresh culture medium, and subculturing according to the proportion of 1:3-1:5.

Feeder cell preparation the procedure for preparing feeder cells using mouse fetal fibroblasts was consistent with that described in example 1.

Example 3: determination of in vitro establishment condition of sheep embryo stem cells

The construction was carried out using three published ESCs culture systems of NBFR (Soto et al 2021), bEPSCM (Zhao et al 2021), 3i/LAF (Zhi et al 2022) and TePR culture systems (based on mTeSR PLUS as basic culture medium supplemented with Wnt signal pathway inhibitor IWR-1) obtained by autonomous modification of CTFR (Vilarino et al 2020) culture systems, respectively (FIGS. 1, 2).

The invention adopts a full embryo inoculation method to construct ESCs, and the specific operation is as follows:

(1) Changing feeder cell culture solution prepared one day in advance, changing into ESCs culture solution, adding 500 μl/well culture solution into 4-well plate, and adding Y-27632 with final concentration of 10 μM;

(2) Placing the blastula into an acidic table fluid, repeatedly blowing the embryo with a mouth suction tube under a split type lens, and removing the transparent belt;

(3) Transferring the blastula with the transparent band removed into a dish containing preheated ESCs culture solution, and washing for 2-3 times;

(4) Transferring blastula into culture solution containing Y-27632, and transferring to 5% CO at 37deg.C ₂ Culturing for 48 hours in an incubator;

(5) The culture medium was gently discarded with a pipette on day 3, and fresh culture medium containing Y-27632 was added, followed by daily replacement of ESCs culture medium containing Y-27632 until cell clones developed;

(6) After the clones were grown to a certain size, the culture solution was discarded, 200. Mu.L of TrypLE select was added, incubated at 37℃for 3min, gently beaten with a pipetting gun, the cell suspension was transferred into a 1.5mL centrifuge tube, centrifuged at 1000rpm for 3min, the supernatant was discarded, the cells were resuspended with fresh ESCs culture solution, and inoculated onto freshly prepared feeder cells in a 1:1 ratio, Y-27632 was added at a final concentration of 10. Mu.M on the day of inoculation, and the ESCs culture solution without Y-27632 was replaced the next day (see FIG. 2 for isolated sESCs).

The results show that when the mouse fetal fibroblasts are used as feeder cells, clear-edge cell clones can be obtained under 4 culture conditions, the morphology is consistent with that of reported sheep and ESCs of other species (figure 2), the obtained cells are respectively named as NBFR-sESCs, bEPSCM-sESCs, 3i/LAF-sESCs and TePR-sESCs (figure 2), but after multiple cell passages (more than 10 generations), the proliferation of the three cells of NBFR-sESCs, bEPSC-sESCs and 3iLAF-sESCs is slow, and then the cells gradually die (figure 3), so that a certain difference exists between embryo stem cell separation culture systems of different species, and the culture conditions of the bovine and porcine embryo stem cells are not suitable for sheep embryo stem cell separation and culture. TePR-sESCs can stably grow in passage. In the case of feeder cells, which were foetal fibroblasts, no obvious cell clones appeared (FIG. 4).

Example 4: sheep embryo stem cell line establishment

1. In vitro isolation of sheep embryo stem cells

Sheep blasts with good morphology and obvious inner cell mass are selected for ESCs in vitro separation, blasts zona pellucida is removed by using acid desk top liquid (Sigma, T1788-100 mL), then the blasts are respectively placed on mouse fetal fibroblast feeder cells, a TePR culture system (mTeSR PLUS is taken as basic culture liquid, wnt signal path inhibitor IWR-1 is added in a supplementing mode) is used for construction, a whole embryo inoculation method is used for in vitro separation and construction of cells, and specific operations are shown in example 3.

2. Passage of sheep embryo stem cells

(1) When the cell clone grows to a certain size, discarding the original culture solution by a pipetting gun, and washing once by D-PBS;

(2) D-PBS was discarded, 200. Mu.L of TrypLE Select was added to each well of the 4-well plate to digest the cells, and after the TrypLE Select was added, the plates were incubated in a 37℃incubator for 3min;

(3) The cells were gently blown with a pipetting gun, the cell suspension was transferred to a 1.5mL centrifuge tube, centrifuged at 1000rpm for 3min, the supernatant was discarded, the cells were resuspended with ESCs broth, blown evenly to avoid large cell clumps, and the cells were seeded onto feeder cells prepared in advance. And Y-27632 was added at a final concentration of 10. Mu.M;

the following day and thereafter the culture medium without Y-27632 is changed daily, and the cells are typically passaged once for 3-4 days.

3. Cryopreservation and resuscitation of sheep embryo stem cells

(1) Cryopreservation of sheep ESCs:

after digesting the cells, collecting cell suspension, centrifuging at 1000rpm for 3min, discarding supernatant, re-suspending the cells with 900 mu L of sESCs culture solution at a ratio of 9:1, transferring the cell suspension into a freezing tube, adding 100 mu L of DMSO, blowing and mixing uniformly by a pipetting gun, marking the freezing tube, placing the frozen tube in a gradient freezing box, freezing in a refrigerator at-80 ℃, and transferring the frozen tube into liquid nitrogen for long-term storage after 24 h.

(2) Recovery of sheep ESCs:

preheating a 37 ℃ water bath, taking out cells to be revived from a liquid nitrogen tank, rapidly placing the cells in the water bath, shaking a freezing tube until the cells are completely dissolved, transferring the liquid in the freezing tube into a 1.5mL centrifuge tube, centrifuging at 1000rpm for 3min, discarding the supernatant, adding fresh sESCs culture solution, resuspending the cells, inoculating the cells onto feeder cells prepared in advance, adding Y-27632 with the final concentration of 10 mu L, changing the culture solution to the culture solution without Y-27632 the next day, and changing the culture solution every day.

Example 5: sheep embryo stem cell identification

1. Morphology observation of sheep embryo stem cells and Alkaline Phosphatase (AP) staining

Firstly, the morphology of the obtained TePR-sESCs is observed, and the obtained TePR-sESCs have smaller cell diameter, large cell nucleus, obvious nucleolus, less cytoplasm, larger nuclear cytoplasm, dome-shaped (domed) growth, clear cell cloning edge, smoother surface, light refraction andthe morphology of the ESCs in the state was consistent and did not change significantly after multiple passages, see FIG. 5.

When the culture confluence of the sheep embryo stem cells reaches 70% -80%, 4% Paraformaldehyde (PFA) is fixed for 1min, the culture is strictly carried out according to the operation instruction of an alkaline phosphatase staining kit (Biyun, P0321S), and the culture is incubated at room temperature for about 15min in a dark place for microscopic examination. The detection results are shown in FIG. 3, and the AP staining results show that TePR-sESCs are positive in alkaline phosphatase staining.

Proliferation potency assay of TePR-sESCs

Sheep Fetal Fibroblasts (SFF) and TePR-sESCs were seeded 3-fold in each 12-well plate, and cells were collected after 3d of incubation and tested using a cell cycle kit, following the kit protocol. The results of the analysis are shown in FIG. 6, which shows that the TePR-sESCs cell line has significantly higher cell numbers in the G2 and S phases than in sheep fibroblasts (P < 0.01).

Analysis of expression of the pluripotent marker Gene of TePR-sESCs

Total RNA of sheep embryo stem cells P10 and P48 were extracted, respectively, and hydrolyzed with RNase-Free water. After measuring the concentration and purity of RNA, a cDNA library is synthesized by reverse transcription, and expression of sheep embryo stem cell multipotent genes OCT4, SOX2 and NANOG is detected by qRT-PCR. As shown in FIG. 7, the mRNA expression levels of the pluripotent genes OCT4, SOX2 and NANOG were significantly higher than those of SFF in the low-algebraic (P10) and high-algebraic (P50) sheep embryo stem cells.

When the confluence of TePR-sESCs reaches about 80%, cells are fixed by 4% PFA for 10min, and then immunofluorescent staining is carried out on the cells, so that the expression of multipotent genes OCT4, SOX2 and NANOG is detected. As shown, OCT4, SOX2 and NANOG proteins were expressed normally (see FIG. 8), and cell surface markers SSEA4, TRA-1-60 and TRA-1-81 were expressed normally (see FIG. 9).

Chromosome number and in vitro differentiation ability analysis of TePR-sESCs

Karyotyping of TePR-sESCs was performed to identify whether chromosomal variation occurred. The results showed that the number of cell chromosomes was normal, 2n=54 (see fig. 10 a), and could be used for subsequent experiments.

And (3) the ESCs are digested into single cells, inoculated into a U-shaped culture dish with low adsorption density according to a certain density, and subjected to suspension culture for 7d, and the liquid is changed every other day. After 7d, the embryoid bodies formed were transferred to gelatin-coated cell culture plates for continuous culture for 14-28d, and as a result, as shown in FIG. 11, the cells were able to form embryoid bodies having a spherical structure, and after in vitro differentiation, qRT-PCR detection was performed on the three germ layer marker genes, respectively, and TePR-sESCs were found to have potential for in vitro differentiation toward three germ layers (FIG. 10b, FIG. 11).

5. In vivo differentiated teratoma

To further verify the differentiation potential of TePR-sESCs, tePR-sESCs were injected subcutaneously into immunized mice, teratomas were formed 10 weeks after injection, and after removal of teratomas, analysis by immunohistochemistry indicated that the formed teratomas had a three-germ layer structure (fig. 12).

Reference is made to:

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

(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.2020 Nov；160(5):761-772.

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

(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 X.Establishment of bovine expanded potential stem cells.Proc Natl Acad Sci U S A.2021 Apr 13；118(15):e2018505118.

(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.

while preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (8)

1. A culture system for improving sheep embryo stem cell multipotency related gene expression is characterized in that the culture system takes mouse fetal fibroblasts as feeder cells and mTESR ^TM Plus-based cultureLiquid, and add 2.5. Mu.M IWR-1 as ESCs culture liquid;

the genes include OCT4, SOX2, NANOG, DPPA3, LIN28, and SALL4.

2. A method of culturing sheep embryo stem cells using the system of claim 1, wherein the sheep embryo stem cells are co-cultured with the culture system.

3. The method according to claim 2, characterized in that it comprises in particular the following steps:

preparing feeder cells;

separating sheep embryo stem cells;

and (5) subculturing sheep embryo stem cells.

4. A method according to claim 3, wherein the feeder cells are prepared by the steps of:

when the mouse fetal fibroblast is cultured until the cell confluence reaches more than 75%, the method comprises the following steps of 1:5, carrying out cell passage according to the proportion;

after the cells grow fully again after passage, the culture solution is replaced by the culture solution containing 10 mug/mL mitomycin C, and the culture solution is treated for 3 hours;

discarding the culture solution containing mitomycin C, washing, and then digesting with 0.25% pancreatin to obtain a cell suspension;

diluting the obtained cell suspension;

the diluted cell suspension is sequentially treated at 4 ℃ for 20min, 20 ℃ for 40min and 80 ℃ for 24h and then transferred into liquid nitrogen for preservation.

5. The method according to claim 4, wherein the method for separating sheep embryo stem cells is a whole embryo seeding method, and comprises the following steps:

and (3) selecting sheep blasts with good morphology and obvious inner cell clusters for ESCs in-vitro separation, and removing blastula zona pellucida by using an acidic desk type liquid.

6. The method according to claim 5, characterized in that the subculture comprises in particular the following steps:

the sheep embryo stem cell clone removes the original culture solution, and the sheep embryo stem cell clone is washed once with D-PBS;

discarding the D-PBS, digesting the cells with TrypLE Select, incubating at 37 ℃ for 3min, and stopping digestion with ESCs culture medium added with Y-27632;

the ESCs culture without Y-27632 was changed the next day and thereafter, and passaged once for 3-4 days.

7. A method for constructing a teratoma animal model, characterized in that the method of claim 5 is used for culturing sheep embryo stem cells, and the obtained sheep embryo stem cells are injected into the skin of a mouse to form teratomas after 10 weeks.

8. Use of an animal model constructed by the method of claim 7 for screening teratoma treatment drugs.