Goat embryonic stem cell culture system, establishment method and application thereof

Abstract

The present application relates to the technical field of cell biology, and specifically discloses a goat embryonic stem cell culture system, a method for establishing a cell line and application, wherein the goat embryonic stem cell culture system is based on mTeSR TM Plus as a basic culture solution, and 20ng / mL-25ng / mL leukemia inhibitory factor, 1M-1.5M PD0325901, 4M-5M IWR-1, 45g / mL-50g / mL Vc, 15ng / mL-20ng / mL activin A, 1-1.2M CGP77675 and 1.5-2M GO6983 are added. The present application can obtain cell clones with clear edges by culturing the goat embryonic stem cell culture system, and the cell clones can be stably passaged and have the potential to differentiate into three germ layers.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell biology, in particular to a goat embryonic stem cell culture system, a method for establishing a cell line and an application thereof. BACKGROUND

[0002] Embryonic stem cells, abbreviated as ESCs, ES, EK or ESC cells, are a kind of cells isolated from early embryos or primordial gonads, which are non-specialized cells, have the characteristics of unlimited proliferation, self-renewal and multi-directional differentiation in vitro culture, and can differentiate into all cell types of an organism.

[0003] The first ESC cell line was first established in mice, and then in human in vitro derived embryos. The methods for establishing and maintaining the ESC cell lines of mice and humans are very mature. In contrast, it is difficult to establish and maintain the embryonic stem cell line from goats due to the difficulty in exploring the culture conditions of goat embryonic stem cells (abbreviated as gESCs) and the low efficiency of deriving ESCs from embryos of livestock species. This may be due to the limited understanding of the biology of ESCs in different species, such as the timing of primary culture and isolation, the culture system that can maintain pluripotency, and the identification of ESCs. At present, the understanding of the biological characteristics, differentiation ability and genetic background of goat in vitro stem cells is still insufficient, and the differences in stem cell research between goats and other animals such as mice or humans may hinder the technical transformation. There is also a bottleneck in the development of in vitro culture conditions, and there is a lack of optimized culture conditions suitable for the growth and differentiation of goat stem cells.

[0004] Most of the existing embryonic stem cell culture systems are derived from other species, such as mouse and human embryonic stem cell culture systems. However, due to the biological differences between species, the use of these embryonic stem cell culture systems in goats can lead to the difficulty in stable passage of goat embryonic stem cells, premature differentiation or death. Therefore, it is of great significance to develop an embryonic stem cell culture system for goats. SUMMARY

[0005] In order to develop a stable and long-term passaged goat embryonic pluripotent stem cell line, the present application provides a goat embryonic stem cell culture system, a method for establishing a cell line and an application thereof. The goat embryonic stem cell culture system of the present application can culture cell clones with clear edges, which can be stably passaged and have the potential to differentiate into three germ layers.

[0006] The present application provides a goat embryonic stem cell culture system, which is mTeSR TMPlus is a basic culture solution, and the following are added: 20ng / mL-25ng / mL leukemia inhibitory factor LIF, 1μM-1.5μM PD0325901, 4μM-5μM IWR-1, 45μg / mL-50μg / mL vitamin C, 15ng / mL-20ng / mL activin A, 1-1.2μM CGP77675, 1.5-2μM GO6983.

[0007] The goat embryonic stem cell culture system provided by the application can successfully isolate goat embryonic stem cells, the isolated goat embryonic stem cells can form clear-edged dome-shaped cell clones, the cell morphology does not change obviously after multiple passages, the pluripotency-related genes remain stable expression, the karyotype is normal, the alkaline phosphatase staining is also positive, and the potential of differentiation into three germ layers is shown.

[0008] Further, the goat embryonic stem cell culture system is mTeSR TM Plus is a basic culture solution, and the following are added: 20ng / mL-25ng / mL leukemia inhibitory factor LIF, 1μM-1.5μM PD0325901, 4μM-5μM IWR-1, 45μg / mL-50μg / mL vitamin C, 15ng / mL-20ng / mL activin A, 1-1.2μM CGP77675, 1.5-2μM GO6983.

[0009] The application further provides a goat embryonic stem cell establishment method, comprising the following steps:

[0010] Preparation of feeder cells: mouse fetal fibroblasts are isolated and subcultured, and then treated with a mouse fetal fibroblast culture solution containing 10μg / mL-15μg / mL mitomycin C for 3h-4h, followed by digestion treatment to obtain mouse fetal fibroblasts as feeder cells;

[0011] Isolation of goat embryonic stem cells by whole embryo inoculation method: the goat embryonic stem cells are isolated by using the culture system of claim 1, and then the isolated goat embryonic stem cells are inoculated on the feeder cells at a ratio of 1:1-3 to obtain goat embryonic stem cells;

[0012] The obtained goat embryonic stem cells are subcultured to obtain a goat embryonic stem cell line;

[0013] The 50mL mouse fetal fibroblast culture solution containing 10μg / mL-15μg / mL mitomycin C has the following formula: 45mL DMEM high-sugar culture medium+5mL fetal bovine serum+500μL penicillin-streptomycin double antibody+10μg / mL-15μg / mL mitomycin C.

[0014] Further, the mouse fetal fibroblast cell culture solution for treating the mouse fetal fibroblast cells in the preparation of the feeder layer cells contains 10 μg / mL of mitomycin C.

[0015] Further, the preparation of the feeder layer cells comprises the following steps:

[0016] When the mouse fetal fibroblast cells are cultured in the mouse fetal fibroblast cell culture solution to a confluence of 75% or above, the cells are subcultured at a ratio of 1:4-5;

[0017] After the subculture, the mouse fetal fibroblast cells completely cover the surface of the culture dish again, the mouse fetal fibroblast cell culture solution is replaced with a culture solution containing 10 μg / mL-15 μg / mL of mitomycin C to treat the cells, and the treatment lasts for 3 hours;

[0018] The mouse fetal fibroblast cell culture solution containing 10 μg / mL-15 μg / mL of mitomycin C is discarded, the mouse fetal fibroblast cells are washed with D-PBS, and then the mouse fetal fibroblast cells are digested with 0.25% trypsin to obtain a mouse fetal fibroblast cell suspension;

[0019] The mouse fetal fibroblast cell suspension containing DMSO after dilution is sequentially placed at 4°C for 20 minutes, at -20°C for 40 minutes, and at -80°C for 24 hours, and then transferred to liquid nitrogen for storage.

[0020] Further, the process for isolating goat embryonic stem cells by whole embryo inoculation is as follows:

[0021] The goat blastocyst with a good morphology and a clear inner cell mass is selected for in vitro isolation of goat embryonic stem cells, the zona pellucida of the blastocyst is removed by using an acidic table solution, and the goat embryonic stem cells are cultured and isolated by using the culture system according to claim 1.

[0022] Further, the subculture process of the goat embryonic stem cells is as follows:

[0023] The goat embryonic stem cells are removed from the original culture solution, D-PBS is added, and the cells are gently washed;

[0024] The D-PBS is discarded, the cells are digested with Accutase, incubated at 37°C for 3 minutes, and then terminated with the gESCs culture solution, and after centrifugation, the supernatant is discarded;

[0025] The cells are resuspended in the goat embryonic stem cell culture medium containing 10 μM-15 μM of Y-27632 at a final concentration, and subcultured at a ratio of 1:3-10 according to the cell growth density;

[0026] The goat embryonic stem cell culture solution without Y-27632 is replaced every 24 hours, and the next passage is performed when the cell confluence reaches 80% to 90%.

[0027] The application further provides a method for constructing a teratoma animal model, wherein the goat embryonic stem cell line is cultured by the method for constructing a goat embryonic stem cell line, 1x10 6 to 2x10 6 cells are resuspended with 200 μL to 300 μL Matrigel and injected subcutaneously into a mouse, and a teratoma is formed after 9 to 10 weeks

[0028] Further, the teratoma has ectoderm, mesoderm and endoderm.

[0029] The application further provides an application of the teratoma animal model in screening of teratoma treatment drugs, wherein the teratoma animal model is constructed by the method for constructing a teratoma animal model.

[0030] Compared with the prior art, the application has the following beneficial effects:

[0031] The gESCs obtained by the culture system and method provided by the application can form a round-topped cell clone with a clear edge. After multiple passages, the morphology of the cells has no obvious change, the pluripotency-related genes are stably expressed, the karyotype is normal, the alkaline phosphatase staining is positive, and the cells show the potential of differentiation into three germ layers.

[0032] The application provides a more suitable environment for the growth and differentiation of goats by innovative culture conditions, thereby solving the problem that goat embryonic stem cells are difficult to stably passaged, prematurely differentiated or die. The culture system and method provided by the application can significantly improve the proliferation and pluripotency of goat embryonic stem cells. The culture system of the application improves the culture efficiency and application potential of goat embryonic stem cells by providing more suitable conditions for the growth and differentiation of goats, and provides an important technical platform for the research and application of goat embryonic stem cells. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0034] Figure 1 The figure is a flow chart of the in-vitro construction of the goat embryonic stem cell line.

[0035] Figure 2The gESCs cell map obtained by the method of Example 2 of the present application,

[0036] In the figure, A is the gESCs cell morphology map after passage, wherein, from left to right, the gESCs cell morphology maps obtained from the second generation, the sixth generation and the tenth generation are shown, and the scale is 1000 μm;

[0037] B is the alkaline phosphatase staining microscopic examination map of the goat embryonic stem cells when the culture confluence reaches 70%, and the scale is 200 μm.

[0038] Figure 3 The cell cycle detection results, * represents significant difference, P < 0.05; ** represents extremely significant difference, P < 0.01; ns represents no significant difference, P > 0.05. Each group of tests is repeated three times;

[0039] In the figure, A is the cell cycle detection result of goat embryonic stem cells (gESCs);

[0040] B is the cell cycle detection result of GFF.

[0041] Figure 4 The cell pluripotency gene expression detection results of GFF, the tenth generation of goat embryonic stem cells (gESCs) and goat fibroblasts (GFF); * represents significant difference, P < 0.05; ** represents extremely significant difference, P < 0.01; ns represents no significant difference, P > 0.05, each group of tests is repeated three times.

[0042] Figure 5 The gESCs, OCT4, SOX2, NANOG cell immunofluorescence staining pictures (scale: 200 μm);

[0043] In the figure, A is the gESCs pluripotency marker NANOG immunofluorescence staining map after passage to the 16th generation;

[0044] B is the pluripotency marker OCT4, SOX2 immunofluorescence staining map of gESCs after passage to the 16th generation.

[0045] Figure 6 The gESCs, SSEA4, TRA-1-81, TRA-1-60 cell immunofluorescence staining pictures (scale: 200 μm);

[0046] In the figure, A is the gESCs cell surface marker SSEA4 immunofluorescence staining map after passage to the 16th generation;

[0047] B is the gESCs cell surface marker TRA-1-60 immunofluorescence staining map after passage to the 16th generation;

[0048] C is the immunofluorescence staining chart of gESCs cell surface marker TRA-A-81 which is passaged to 16 generations.

[0049] Figure 7 Figure A is the karyotype of goat embryonic stem cells (gESCs) ;

[0050] Figure A is the karyotype of goat embryonic stem cells (gESCs) ;

[0051] Figure B is a picture of a goat embryonic stem cell (gESCs) embryoid body, wherein the first column from left to right is the immunofluorescence staining of β-Tubulin, α-SMA, Nestin and AFP; the second column from left to right corresponds to the cell nucleus staining of the cells in the first column.

[0052] Figure 8 Figure B is a picture of a goat embryonic stem cell (gESCs) embryoid body, wherein the first column from left to right is the immunofluorescence staining of β-Tubulin, α-SMA, Nestin and AFP; the second column from left to right corresponds to the cell nucleus staining of the cells in the first column. DETAILED DESCRIPTION

[0053] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0054] Example 1: Preparation of mouse fetal fibroblast feeder cells.

[0055] 1. Isolation of mouse fetal fibroblasts

[0056] (1) The fetal mouse was removed from the 6-8 week old C57BL / 6 female mouse fetus at 12 days of gestation, and then the head, limbs, internal organs and tail were removed using tweezers and scissors, and the remaining tissue was washed in a pre-prepared Dulbecco's phosphate buffer solution;

[0057] (2) The washed tissue was cut into small pieces using scissors to obtain tissue pieces, 5g of tissue pieces were taken and digested with 4mL of trypsin solution at a mass / volume ratio of 0.25% at room temperature for 15min. Then the tissue pieces were blown and dispersed uniformly with a 15mL pipette, and continued to be digested for 5min, then 1mL of mouse fetal fibroblast culture solution was added to complete the digestion process, and a cell suspension was obtained;

[0058] 0.25% trypsin solution was obtained by dissolving 0.25 g trypsin in 100 mL PBS buffer.

[0059] The mouse fetal fibroblast culture solution (50 mL) was formulated as follows: 45 mL DMEM high-sugar medium + 5 mL fetal bovine serum + 500 μL penicillin-streptomycin double antibody.

[0060] (3) The cell suspension in step (2) was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 1 mL of mouse fetal fibroblast culture solution was added to resuspend the cells, and the cells were inoculated into a 100 mm culture dish and cultured in a 37°C, 5% CO2 incubator;

[0061] (4) When the mouse fetal fibroblast cells reached more than 95% confluence, 2 mL of 0.25% trypsin solution was used to digest the cells again for 1 min at 37°C in a 5% CO2 incubator, to obtain a mouse fetal fibroblast cell suspension, and the mouse fetal fibroblast cells were diluted to contain 1×10 6 mouse fetal fibroblast cells per 1 mL using the mouse fetal fibroblast freezing solution, to obtain frozen mouse fetal fibroblast cells, which were ready for use.

[0062] The mouse fetal fibroblast freezing solution (10 mL) was formulated as follows: 9 mL mouse fetal fibroblast culture solution + 1 mL DMSO.

[0063] 2. Preparation of feeder layer cells

[0064] (1) The frozen mouse fetal fibroblast cells were thawed, and 1×10 6 mouse fetal fibroblast cells were inoculated into a 100 mm cell culture dish. When the cells reached more than 75% confluence, the cells were subcultured at a ratio of 1:5;

[0065] (2) After the cells completely covered the surface of the culture dish again after subculture, the mouse fetal fibroblast culture solution was replaced with mouse fetal fibroblast culture solution containing 10 μg / mL mitomycin C to treat the cells, and the treatment lasted for 3 h;

[0066] (3) The mouse fetal fibroblast culture solution containing mitomycin C was discarded, the cells were washed with preheated D-PBS 5 times, and then 2 mL of 0.25% trypsin solution was used to digest the cells for 1 min at 37°C in a 5% CO2 incubator, to obtain a feeder layer cell suspension, i.e., mouse fetal fibroblast cells treated with mitomycin C;

[0067] (4) The obtained feeder layer cell suspension was counted, and the cells were diluted to contain 6×10 5 feeder layer cells per 1 mL using the mouse fetal fibroblast freezing solution, and the feeder layer cells were aliquoted into freezing tubes for cell freezing.

[0068] (5) The sub-packaged feeder cells to be frozen are sequentially treated at 4°C for 20 min, -20°C for 40 min, and -80°C for 16 h, and then transferred to liquid nitrogen for preservation.

[0069] Example 2: In vitro establishment of goat embryonic stem cell lines.

[0070] This example uses the whole embryo inoculation method to establish gESCs, and the specific operation is as follows:

[0071] (1) One day in advance, prepare feeder cells for subculture, add 500 μL of 0.1% gelatin aqueous solution to a 4-well plate, incubate at 37°C for 30 min, and then discard the 0.1% gelatin aqueous solution; add feeder cells at a cell amount of 6 x 10 5 / well to the 4-well plate, and place the culture plate in a 37°C incubator for 6 h.

[0072] (2) Discard the feeder cell culture supernatant in the 4-well plate, wash once with D-PBS, replace with gESCs culture medium, add 500 μL / well of gESCs culture medium to the 4-well plate, and add Y-27632 at a final concentration of 10 μM;

[0073] The gESCs culture medium is: mTeSR TM Plus as the basic culture medium, supplemented with leukemia inhibitory factor at a final concentration of 25 ng / mL, PD0325901 at 1 μM, IWR-1 at 5 μM, Vc at 50 μg / mL, Activin A at 20 ng / mL, CGP77675 at 1.2 μM, and GO6983 at 2 μM.

[0074] (2) Select a goat blastula with good morphology and obvious inner cell mass from the goat uterus, place it in acidic table liquid, and under a stereomicroscope, repeatedly blow the embryo with a mouth pipette to remove the zona pellucida;

[0075] (3) Transfer the zona pellucida-removed blastula to a culture dish containing preheated gESCs culture medium, and wash 3 times with preheated gESCs culture medium;

[0076] (4) Transfer the blastula to gESCs culture medium supplemented with Y-27632 at a final concentration of 10 μM, and culture in a 37°C, 5% CO2 incubator for 48 h;

[0077] (5) On the 3rd day, gently discard the culture medium supplemented with Y-27632 with a pipette, and then replace it with gESCs culture medium without Y-27632 every 24 h until cell clones grow out;

[0078] (6) When the gESCs cell clones grow to 80% confluence, discard the gESCs culture solution, add 200 μL of Accutase enzyme, incubate at 37°C for 3 min, obtain the gESCs cell suspension, gently blow with a pipette and transfer the gESCs cell suspension to a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, collect the cell pellet, resuspend the cell pellet with fresh gESCs culture solution, inoculate the gESCs on the feeder layer cells prepared in step (1), and culture in a 37°C 5% CO2 incubator for 3 days, add Y-27632 with a final concentration of 10 μM on the inoculation day, and replace the gESCs culture solution without Y-27632 on the second day, to obtain the goat embryonic stem cell line in vitro.

[0079] The results show that when the mouse fetal fibroblasts are used as feeder layer cells and the gESCs culture solution is used for culture, cell clones with clear edges can be obtained under the culture condition, and the morphology is consistent with the morphology of reported sheep and other species ESCs. Figure 2 The obtained cells are named gESCs. Figure 2

[0080] Example 3: Goat embryonic stem cell line construction

[0081] 1. In vitro isolation of goat embryonic stem cells

[0082] Select the goat blastocyst with good morphology and obvious inner cell mass from the goat uterus for in vitro isolation of gESCs. After removing the zona pellucida of the blastocyst with acid table solution, place it on the feeder layer cells, i.e. mouse fetal fibroblasts, and use the 4i-LA culture system for line construction. The whole embryo inoculation method is used for in vitro isolation and line construction. The specific operation is referred to Example 2, and the goat embryonic stem cell line is obtained.

[0083] The 4i-LA culture solution (i.e. gESCs culture solution) is based on mTeSR TM Plus culture solution, supplemented with 25 ng / mL leukemia inhibitory factor, 1 μM PD0325901, 5 μM IWR-1, 50 μg / mL Vc, 20 ng / mL activin A, 1.2 μM CGP77675, and 2 μM GO6983.

[0084] 2. Passage of goat embryonic stem cells

[0085] (1) When the gESCs cell clones grow to 80% confluence, prepare the feeder layer cells in advance for passage one day in advance, treat the 4-well plate with 0.1% gelatin aqueous solution, incubate at 37°C for 30 min, and discard the 0.1% gelatin aqueous solution; add 6×10 5 ​ / well feeder cells, and culture plate in 37℃ incubator for more than 6 hours.

[0086] (2) On the day of passage, the goat embryonic stem cell line was discarded from the gESC culture medium. 200 μL of Accutase enzyme was added to each well of a 4-well plate to digest the cells. After adding the Accutase enzyme, the culture plate was placed in a 37°C incubator for 3 min.

[0087] (3) Gently pipette the cells to obtain a gESCs cell suspension. Transfer the gESCs cell suspension to a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, collect the gESCs cell pellet, resuspend the cell pellet in gESCs culture medium, and gently pipette to avoid large cell clumps. Then, seed the cells onto a pre-prepared feeder cell layer and add Y-27632 to a final concentration of 10 μM and continue culturing for 24 h. Afterward, replace the gESCs culture medium without Y-27632 every 24 h. When the cell confluence reaches 80%, the next passage can be performed.

[0088] 3. Cryopreservation and thawing of goat embryonic stem cells

[0089] (1) Cryopreservation of goat ESCs:

[0090] On the day of cryopreservation, discard the gESC culture medium. Add 200 μL Accutase enzyme to each well of a 4-well plate to digest the cells. After adding the Accutase enzyme, incubate the culture plate at 37°C for 3 min. Gently pipette the cells to collect the gESC cell suspension. Centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells in gESC cryopreservation medium, mix well by pipetting, and transfer to cryopreservation tubes. Label the cryopreservation tubes and place them in a gradient cryopreservation box at -80°C. After 24 h, transfer to liquid nitrogen for long-term storage.

[0091] (2) ESCs recovery in goats

[0092] Taking a 4-well plate as an example, goat ESCs resuscitation was performed.

[0093] a. Prepare feeder cells one day in advance for passage. Treat 4-well plates with a 0.1% gelatin aqueous solution, incubate at 37°C for 30 min, then discard. Add cells to the 4-well plates at a rate of 6 × 10⁶ cells / well. 5 Feeder cells per well were cultured in a 37°C incubator for at least 6 hours.

[0094] b. Preheat 37℃ water bath, take the frozen gESCs from the liquid nitrogen tank and put it into the water bath immediately, shake the frozen tube until it completely dissolved, transfer the liquid in the frozen tube to a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells with fresh gESCs culture medium, inoculate the prepared 4-well plate feeder layer cells in the previous step, and add Y-27632 with a final concentration of 10 μM for continuous culture for 24 h; Then replace the gESCs culture medium without Y-27632 every 24 h, and when the cell confluence reaches 80%, the next passage can be carried out.

[0095] Example 4: Identification of goat embryonic stem cells.

[0096] 1. Morphological observation of goat embryonic stem cells and alkaline phosphatase staining

[0097] First, the morphology of the obtained gESCs was observed, and it was found that the gESCs had a small cell diameter, a large nucleus, a clear nucleolus, a small cytoplasm, a large nucleolus, a round top growth, a clear cell clone edge, a smooth surface, and a refraction, which was consistent with the morphology of the original gESCs, and the morphology did not change significantly after multiple passages, as shown in Figure 2 A.

[0098] When the confluence of the goat embryonic stem cells reached 70%, 4% paraformaldehyde was used for fixation for 1 min, and strict operation according to the operation instruction of the alkaline phosphatase staining kit was carried out, incubation at room temperature for 15 min in the dark, and microscopic examination. The detection results are shown in Figure 2 B, and the AP staining results showed that the gESCs were positive for alkaline phosphatase staining.

[0099] 2. Analysis of the proliferation ability of goat embryonic stem cells

[0100] Inoculate goat fetal fibroblasts and goat embryonic stem cells into 12-well plates, 3 repeats each, collect the cells after 3 days of culture, and use the cell cycle kit for detection, according to the steps in the kit instruction manual. The analysis results are shown in Figure 3 It is shown that compared with goat fibroblasts, the number of cells in G1 and S phase of the goat embryonic stem cell line is significantly higher than that of goat fetal fibroblasts, P<0.01.

[0101] 3. Expression analysis of pluripotency marker genes of goat embryonic stem cells

[0102] Total RNA was extracted from goat embryonic stem cells (gESCs) cultured under 4i / LA conditions and from goat fetal fibroblasts (GFFs) cultured under fibroblast conditions, and hydrolyzed with nuclease-free water. After measuring the concentration and purity of the RNA, cDNA libraries were synthesized via reverse transcription, and the expression levels of OCT4, SOX2, NANOG, KLF4, DPPA3, SALL4, and REX1 were analyzed. Results are as follows: Figure 4 As shown, compared with GFF cells, the mRNA expression levels of pluripotency genes OCT4, SOX2, and NANOG in 4i / LA-gESCs were significantly higher than those in GFF cells (P<0.05).

[0103] NANOG is a transcription factor containing a homologous domain, essential for maintaining the pluripotency and self-renewal of embryonic stem cells. OCT4 is a transcription factor expressed in undifferentiated pluripotent embryonic stem cells and germ cells during normal development. SOX2 is a transcription factor expressed in undifferentiated pluripotent embryonic stem cells and germ cells during development.

[0104] When gESC confluence reached approximately 80%, the cells were fixed with 4% PFA for 10 min, followed by immunofluorescence staining 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 5 Cell surface markers SSEA4, TRA-1-60, and TRA-1-81 are normally expressed (see [link to relevant documentation]). Figure 6 ).

[0105] The cell surface marker SSEA4 is a glycolipid carbohydrate expressed on the surface of human teratoma stem cells, embryonic germ cells, and embryonic stem cells. Cell surface markers TRA-1-60 and TRA-A-81 are present on the surface of human stem cells, teratoma cells, and embryonic germ cells.

[0106] 4. Analysis of chromosome number and in vitro differentiation capacity of gESCs

[0107] Karyotype analysis was performed on gESCs to identify whether chromosomal aberrations occurred. Results showed that the cell chromosome number was normal, 2n = 60 (see...). Figure 7 A) can be used for subsequent experiments.

[0108] gESCs were digested into single cells and seeded at a certain 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 28 days. The results are as follows: Figure 7As shown in FIG. 4B, cells were able to form embryoid bodies with spherical structure. Immunofluorescence staining was performed on cells after in vitro differentiation. In the figure, β-Tubulin is a cytoskeletal protein, tubulin marker, which is a mesoderm and ectoderm related marker; α-SMA is a fibroblast activation marker, which is a mesoderm marker; Nestin is an intermediate filament protein marker expressed in neural stem cells and various nervous system tumor cells, which is a neuroectoderm marker; AFP is an endodermal layer marker. The results show that mesoderm marker proteins β-Tubulin and α-SMA, ectoderm marker protein Nestin, and endoderm marker protein AFP are normally expressed (see Figure 7 ). Subsequently, RT-qPCR detection was performed on three germ layer marker genes, respectively, and the results showed that each germ layer marker gene was significantly expressed (see Figure 8 )(P<0.05). It is proved that 4i / LA-gESCs cultured in the 4i / LA system proposed by the application have the potential to differentiate into three germ layers in vitro.

[0109] 5. In vivo differentiation teratoma

[0110] To further verify the differentiation potential of gESCs, gESCs were injected into immunodeficient mice subcutaneously. After 10 weeks of injection, teratomas were formed. Subsequently, the teratomas were removed and subjected to immunohistochemical analysis, and the results showed that the formed teratomas had three germ layer structures.

[0111] The Chinese names of the English terms involved in the application are listed in Table 1.

[0112] Table 1: English term comparison table

[0113] Serial number Chinese name English name 1 Dulbecco's phosphate buffered saline D-PBS 2 Activin A Activin A 3 Goat embryonic stem cells gESCs 4 Alkaline phosphatase AP 5 Polyformaldehyde PFA 6 Goat fetal fibroblasts GFF 7 Rnase-free water Rnase-free water Name Brand and specification 1 Acidic table solution Sigma, T1788-100mL 2 Alkaline phosphatase staining kit Bi Yun Tian, P0321S

[0114] Although the preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic inventive concept.

[0115] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application belong to the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A method for establishing a line of goat embryonic stem cells, characterized by, The method comprises the following steps: Preparation of feeder cells: mouse fetal fibroblasts are isolated and cultured in mouse fetal fibroblast culture medium until the cell confluence reaches more than 75%, and then the cells are subcultured at a ratio of 1:4-5; After the subculture, the mouse fetal fibroblasts completely cover the surface of the culture dish again, and the mouse fetal fibroblast culture medium is replaced with mouse fetal fibroblast culture medium containing 10 μg / mL mitomycin C to treat the cells, and the treatment time is 3 h; The mouse fetal fibroblast culture medium containing 10 μg / mL mitomycin C is discarded, the mouse fetal fibroblasts are washed with D-PBS, and then the mouse fetal fibroblasts are digested with 0.25% trypsin to obtain a mouse fetal fibroblast suspension; The diluted mouse fetal fibroblast suspension containing DMSO is sequentially placed at 4°C for 20 min, -20°C for 40 min, and -80°C for 24 h, and then transferred to liquid nitrogen for storage; Isolation of goat embryonic stem cells by whole embryo inoculation: select goat blastocysts with good morphology and obvious inner cell mass for in vitro isolation of goat embryonic stem cells, remove the zona pellucida of the blastocyst using acid table liquid, and isolate goat embryonic stem cells using a goat embryonic stem cell culture system, then inoculate the isolated goat embryonic stem cells into the feeder layer cells at a ratio of 1:1-3 to obtain goat embryonic stem cells; The goat embryonic stem cell culture system is based on mTeSR TM Plus as the basic culture solution, and adding: 20 ng / mL-25 ng / mL leukemia inhibitory factor LIF, 1 µM-1.5 µM PD0325901, 4 µM-5 µM IWR-1, 45 µg / mL-50 µg / mL vitamin C, 15 ng / mL-20 ng / mL activin A, 1-1.2 µM CGP77675, 1.5-2 µM GO6983; Subculture the obtained goat embryonic stem cells: remove the original culture medium of the goat embryonic stem cells, add D-PBS, and gently shake to wash the cells; Discard the D-PBS, digest the cells with Accutase, incubate at 37°C for 3 min, terminate the digestion with gESCs culture medium, centrifuge and discard the supernatant; Resuspend the cells in goat embryonic stem cell culture medium containing a final concentration of 10 μM-15 μM Y-27632, and subculture at a ratio of 1:3-10 according to the cell growth density; Change the goat embryonic stem cell culture medium without Y-27632 every 24 h, and subculture when the cell confluence reaches 80%-90% for the next time to obtain a goat embryonic stem cell line; The formula of 50 mL of the mouse fetal fibroblast culture medium containing 10 μg / mL mitomycin C is: 45 mL DMEM high-sugar medium + 5 mL fetal bovine serum + 500 μL penicillin-streptomycin double antibody + 10 μg / mL mitomycin C.

2. A method for constructing a teratocarcinoma animal model, characterized by, Using the method described in claim 1 to culture goat embryonic stem cells, 1x10 6 2 x 10 6 A number of goat embryonic stem cells were resuspended in 200-300 μL of matrix gel and injected subcutaneously into mice. After 9-10 weeks, teratomas were formed.

3. The method of constructing a teratoma animal model according to claim 2, wherein The teratoma has ectoderm, mesoderm and endoderm. The teratoma has ectoderm, mesoderm and endoderm.

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