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

Abstract

The invention relates to the technical field of cell biology, and particularly discloses a goat embryonic stem cell culture system, a system establishment method and application, the goat embryonic stem cell culture system takes mTeSRTM Plus as a basic culture solution, and 20 ng / mL to 25 ng / mL of leukemia inhibition factors, 1 mu M to 1.5 mu M of PD0325901, 4 mu M to 5 mu M of IWR-1, 45 mu g / mL to 50 mu g / mL of Vc, 15 ng / mL to 20 ng / mL of activin A, 1 mu M to 1.2 mu M of CGP77675 and 1.5 mu M to 2 mu M of GO6983 are added. Cell clone with clear edges is obtained through culture of a goat embryonic stem cell culture system, stable passage can be achieved, and the cell clone has the potential of being differentiated into three germ layers.

Description

Technical Field

[0001] The invention relates to the technical field of cell biology, and in particular to a goat embryonic stem cell culture system and a system establishment method and application. Background Art

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

[0003] The first ESCs cell line was first established in mice and then in human embryos derived in vitro. The methods for establishing and maintaining mouse and human ESCs cell lines are well established. In contrast, goat embryonic stem cells, or gESCs, have been difficult to establish and maintain due to the difficulty in exploring the establishment and culture conditions of goat embryonic stem cells, and the low efficiency of deriving ESCs from the embryos of livestock species. This may be due to the limited understanding of the biology of ESCs in different species, such as the timing and isolation of primary cultures, culture systems that can maintain pluripotency, and identification of ESCs. At present, the understanding of the biological characteristics, differentiation capacity, and genetic background of goat in vitro stem cells is still insufficient, and the differences in research on stem cells between goats and other animals such as mice or humans may hinder technology translation. There are also development bottlenecks in 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 borrowed from other species, such as mouse and human embryonic stem cell culture systems. However, given the biological differences between species, these embryonic stem cell culture systems are prone to cause goat embryonic stem cells to be difficult to pass on stably, differentiate prematurely or die. Embryonic stem cell culture systems for goats are currently rare, so it is of great significance to develop embryonic stem cell culture systems for goats. Summary of the invention

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

[0006] The present invention provides a goat embryonic stem cell culture system, wherein the goat embryonic stem cell culture system is based on mTeSR TMPlus is the basic culture medium, and is supplemented with: 20ng / mL~25ng / mL of leukemia inhibitory factor LIF, 1μM~1.5μM of PD0325901, 4μM~5μM of IWR-1, 45μg / mL~50μg / mL of vitamin C, 15ng / mL~20ng / mL of activin A, 1~1.2μM of CGP77675, and 1.5~2μM of GO6983.

[0007] The goat embryonic stem cell culture system provided by the present invention can successfully isolate goat embryonic stem cells. The isolated goat embryonic stem cells can form dome-shaped cell clones with clear edges. After multiple rounds of passages, the cell morphology does not change significantly, the pluripotency-related genes remain stably expressed, the karyotype is normal, alkaline phosphatase staining is also positive, and the cells show the potential to differentiate into three germ layers.

[0008] Furthermore, the goat embryonic stem cell culture system is based on mTeSR TM Plus is the basic culture medium, and is supplemented with: 25 ng / mL leukemia inhibitory factor, 1 μM PD0325901, 5 μM IWR-1, 50 μg / mL vitamin C, 20 ng / mL activin A, 1.2 μM CGP77675, and 2 μM GO6983.

[0009] The present invention also provides a method for establishing a goat embryonic stem cell line, comprising the following steps:

[0010] Preparation of feeder layer cells: Mouse fetal fibroblasts were isolated and subcultured, treated with mouse fetal fibroblast culture medium containing 10 μg / mL to 15 μg / mL mitomycin C for 3 h to 4 h, and then digested to obtain mouse fetal fibroblasts as feeder layer cells;

[0011] Whole embryo inoculation method to isolate goat embryonic stem cells: using the culture system described in claim 1 to isolate goat embryonic stem cells, and then inoculating the isolated goat embryonic stem cells into feeder layer cells at a ratio of 1:1 to 3 to obtain goat embryonic stem cells;

[0012] Subculturing the obtained goat embryonic stem cells to obtain a goat embryonic stem cell line;

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

[0014] Furthermore, the mouse fetal fibroblast culture medium used to treat the mouse fetal fibroblasts during the preparation of the feeder layer cells contains 10 μg / mL mitomycin C.

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

[0016] Mouse fetal fibroblasts were cultured in mouse fetal fibroblast culture medium until the cell confluence reached more than 75%, and then the cells were passaged at a ratio of 1:4-5;

[0017] After the subculture, when the mouse fetal fibroblasts completely cover the surface of the culture dish again, the mouse fetal fibroblast culture medium is replaced with a culture medium containing 10 μg / mL to 15 μg / mL mitomycin C and the cells are treated for 3 hours;

[0018] The mouse fetal fibroblast culture medium containing 10 μg / mL to 15 μg / mL mitomycin C was discarded, the mouse fetal fibroblasts were rinsed with D-PBS, and then the mouse fetal fibroblasts were digested with 0.25% trypsin to obtain a mouse fetal fibroblast suspension;

[0019] The diluted mouse fetal fibroblast cell suspension containing DMSO was 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.

[0020] Furthermore, the whole embryo inoculation method is used to isolate goat embryonic stem cells as follows:

[0021] Goat blastocysts with good morphology and obvious inner cell mass are selected for in vitro isolation of goat embryonic stem cells, the zona pellucida of the blastocyst is removed using an acidic benchtop solution, and the culture system described in claim 1 is used to culture and isolate goat embryonic stem cells.

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

[0023] Remove the original culture medium of goat embryonic stem cells and add D-PBS to gently shake and rinse the cells;

[0024] Discard the D-PBS, digest the cells with Accutase, incubate at 37°C for 3 min, terminate the digestion with gESCs culture medium, and discard the supernatant after centrifugation;

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

[0026] During subculturing, the goat embryonic stem cell culture medium without Y-27632 was replaced every 24 hours, and the next subculturing could be performed when the cell confluence reached 80% to 90%.

[0027] The present invention also provides a method for constructing a teratoma animal model, wherein goat embryonic stem cells are cultured using the goat embryonic stem cell establishment method, and 1x10 6 ~2x10 6 Goat embryonic stem cells with a cell number were resuspended in 200 μL to 300 μL of matrix gel and injected into the subcutaneous tissue of mice. After 9-10 weeks, teratomas were formed.

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

[0029] The present invention also provides an application of a teratoma animal model in screening teratoma therapeutic drugs, wherein the teratoma animal model is constructed by the teratoma animal model construction method.

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

[0031] The gESCs cultured using the culture system and method provided by the present invention can form dome-shaped cell clones with clear edges. After multiple rounds of passages, the cell morphology does not change significantly, the pluripotency-related genes remain stably expressed, the karyotype is normal, alkaline phosphatase staining is also positive, and the cells show the potential to differentiate into three germ layers.

[0032] The present invention provides an environment more suitable for goat growth and differentiation through innovative culture conditions, thereby solving the current problems of goat embryonic stem cells being difficult to stably propagate, prematurely differentiate or die. The culture system and method provided by the present invention can significantly improve the proliferation and pluripotency of goat embryonic stem cells. The culture system of the present invention improves the culture efficiency and application potential of goat embryonic stem cells by providing conditions more suitable for goat growth and differentiation, and provides an important technical platform for the research and application of goat embryonic stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The figure is a flow chart of the in vitro establishment of goat embryonic stem cell system of the present invention.

[0035] Figure 2This is a cell diagram of gESCs obtained by the method of Example 2 of the present invention;

[0036] In the figure, A is a cell morphology diagram of gESCs after passage, wherein from left to right, the cell morphology diagrams of gESCs obtained from the second, sixth and tenth generations are shown, scale: 1000 μm;

[0037] B is an alkaline phosphatase staining microscopic examination of goat embryonic stem cells when the confluence of culture reached 70%, scale bar: 200 μm.

[0038] Figure 3 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 experiments was 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 These are the results of pluripotency gene expression in GFF, tenth-generation 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 experiments was repeated three times.

[0042] Figure 5 For gESCs, OCT4, SOX2, and NANOG cell immunofluorescence staining images (scale bar: 200 μm);

[0043] In the figure, A is an immunofluorescence staining image of NANOG, a pluripotency marker, of gESCs passaged to passage 16;

[0044] B is the immunofluorescence staining of pluripotency markers OCT4 and SOX2 of gESCs passaged to passage 16.

[0045] Figure 6 Immunofluorescence staining images of gESCs, SSEA4, TRA-1-81, and TRA-1-60 cells (scale bar: 200 μm);

[0046] In the figure, A is the immunofluorescence staining of SSEA4, a cell surface marker of gESCs passaged to passage 16;

[0047] B is the immunofluorescence staining of TRA-1-60, a cell surface marker of gESCs cultured to passage 16;

[0048] C is the immunofluorescence staining of TRA-A-81, a cell surface marker, in gESCs cultured to passage 16.

[0049] Figure 7 The karyotype and embryoid body diagram of goat embryonic stem cells;

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

[0051] B is an embryoid body image of goat embryonic stem cells (gESCs), in which 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 nuclear staining of the stained cells in the first column.

[0052] Figure 8 These are the results of mRNA gene expression detection in the three germ layers of embryoid bodies; * represents significant difference, P<0.05; ** represents extremely significant difference, P<0.01; ns represents no significant difference, P>0.05. Each group of experiments was repeated three times. DETAILED DESCRIPTION

[0053] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

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

[0055] 1. Isolation of Mouse Fetal Fibroblasts

[0056] (1) Remove the fetus from a 6-8 week old C57BL / 6 female mouse on the 12th day of pregnancy, and then use forceps and scissors to remove its head, limbs, internal organs and tail, and wash the remaining tissue in pre-prepared Dulbecco's phosphate buffered saline;

[0057] (2) Use scissors to cut the cleaned tissue into small pieces to obtain tissue blocks. Take 5 g of tissue blocks and add 4 mL of 0.25% pancreatic enzyme solution at room temperature for 15 minutes. Then use a 15 mL pipette to blow the tissue blocks to make them evenly dispersed. After continuing to digest for 5 minutes, add 1 mL of mouse fetal fibroblast culture medium to complete the digestion process and obtain a cell suspension;

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

[0059] The formula of the mouse fetal fibroblast culture medium (50 mL) is: 45 mL DMEM high-glucose culture medium+5 mL fetal bovine serum+500 μL penicillin-streptomycin double antibody.

[0060] (3) centrifuging the cell suspension in step (2) at 1000 rpm for 5 min, discarding the supernatant, adding 1 mL of mouse fetal fibroblast culture medium to resuspend the cells, and inoculating them into a 100 mm culture dish, and culturing them in a 37° C., 5% CO 2 incubator;

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

[0062] The formula of the mouse fetal fibroblast cryopreservation solution (10mL) is: 9mL mouse fetal fibroblast culture solution+1mL DMSO.

[0063] 2. Preparation of feeder cells

[0064] (1) Thaw frozen mouse fetal fibroblasts and take 1×10 6 Mouse fetal fibroblasts were inoculated in 100 mm cell culture dishes, and when the cell confluence reached more than 75%, the cells were passaged at a ratio of 1:5.

[0065] (2) After the cells completely covered the surface of the culture dish again after passage, the mouse fetal fibroblast culture medium was replaced with a mouse fetal fibroblast culture medium containing 10 μg / mL mitomycin C and the cells were treated for 3 h;

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

[0067] (4) Count the obtained feeder layer cell suspension and dilute the cells to 6×10 cells per 1 mL using mouse fetal fibroblast cryopreservation solution. 5 Feeder layer cells are divided into cryopreservation tubes for cell cryopreservation;

[0068] (5) After packaging, the feeder layer cells to be frozen were 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 storage.

[0069] Example 2: Method for establishing goat embryonic stem cell line in vitro.

[0070] In this example, the whole embryo seeding method was used to establish the gESCs line, and the specific operation was as follows:

[0071] (1) Prepare feeder layer cells for passage one day in advance. Add 500 μL of 0.1% gelatin aqueous solution to a 4-well plate. Incubate at 37°C for 30 min, then discard the 0.1% gelatin aqueous solution. Add 6×10 cells to the 4-well plate. 5 / well feeder layer cells, and place the culture plate in a 37°C incubator for 6 hours.

[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 gESCs culture medium to the 4-well plate, and add Y-27632 with a final concentration of 10 μM;

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

[0074] (2) selecting a goat blastocyst with good morphology and obvious inner cell mass taken from the goat uterus, placing it in an acidic benchtop solution, and repeatedly blowing the embryo with a mouth pipette under a stereomicroscope to remove the zona pellucida;

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

[0076] (4) The blastocysts were transferred to gESCs culture medium supplemented with Y-27632 at a final concentration of 10 μM and cultured in an incubator at 37°C and 5% CO2 for 48 h;

[0077] (5) On the third day, the culture medium supplemented with Y-27632 was gently discarded with a pipette, and then replaced with gESCs culture medium without Y-27632 every 24 h until cell clones grew;

[0078] (6) When the gESCs clones grow to 80% confluence, the gESCs culture medium is discarded, 200 μL Accutase enzyme is added, and the cells are incubated at 37°C for 3 min to obtain a gESCs cell suspension. The gESCs cell suspension is gently blown with a pipette and transferred to a 1.5 mL centrifuge tube. After centrifugation at 1000 rpm for 3 min, the supernatant is discarded, the cell pellet is collected, and the cell pellet is resuspended with fresh gESCs culture medium. The gESCs are inoculated on the feeder layer cells prepared in step (1) and cultured in a 37°C 5% CO2 incubator for 3 days. Y-27632 is added at a final concentration of 10 μM on the day of inoculation, and the culture medium is replaced with a gESCs culture medium without Y-27632 on the second day to obtain an in vitro goat embryonic stem cell line.

[0079] The results showed that when mouse fetal fibroblasts were used as feeder cells and cultured with the gESCs culture medium of the present invention, cell clones with clear edges could be obtained under the culture conditions, and the morphology was consistent with the reported morphology of ESCs of sheep and other species ( Figure 2 ), and the obtained cells were named gESCs ( Figure 2 ).

[0080] Example 3: Establishment of goat embryonic stem cell line.

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

[0082] Goat blastocysts with good morphology and obvious inner cell mass taken out from the goat uterus were selected for in vitro isolation of gESCs. The zona pellucida of the blastocyst was removed using acidic benchtop solution, and then placed on feeder layer cells, i.e., mouse fetal fibroblasts. The line was established using the 4i-LA culture system, and the whole embryo inoculation method was used for in vitro cell isolation and line establishment. The specific operations were referred to Example 2 to obtain a goat embryonic stem cell line.

[0083] The 4i-LA culture medium (i.e., gESCs culture medium) is based on mTeSR TM Plus is the basic culture medium, 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. Passaging of goat embryonic stem cells

[0085] (1) When the gESCs cell clones grow to 80% density, prepare the feeder layer cells 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 then discard the 0.1% gelatin aqueous solution; add 6×10 cells to the 4-well plate. 5 / well feeder layer cells, and place the culture plate in a 37°C incubator for more than 6 hours.

[0086] (2) On the day of subculturing, the goat embryonic stem cell line discarded the gESCs culture medium, added 200 μL Accutase enzyme to each well of a 4-well plate to digest the cells, and after adding Accutase enzyme, placed the culture plate in a 37°C incubator for 3 min;

[0087] (3) Use a pipette to gently blow 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 with gESCs culture medium, blow evenly to avoid large cell clumps, then inoculate the cells on the feeder layer cells prepared in advance, and add Y-27632 with a final concentration of 10 μM to continue culturing for 24 hours. After that, replace the gESCs culture medium without adding Y-27632 every 24 hours, and the next passage can be carried out when the cell confluence reaches 80%.

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

[0089] (1) Cryopreservation of goat ESCs:

[0090] On the day of freezing, discard the gESCs culture medium, add 200 μL Accutase enzyme to each well of the 4-well plate to digest the cells, and after adding Accutase enzyme, place the culture plate in a 37°C incubator for 3 minutes. Use a pipette to gently blow the cells, collect the gESCs cell suspension, centrifuge at 1000rpm for 3 minutes, discard the supernatant, resuspend the cells with gESCs freezing solution, blow and mix with a pipette, and transfer to a cryopreservation tube. After marking the cryopreservation tube, place it in a gradient cryopreservation box and freeze it in a -80°C refrigerator. After 24 hours, transfer it to liquid nitrogen for long-term storage.

[0091] (2) Recovery of goat ESCs

[0092] Take 4-well plates as an example to recover goat ESCs:

[0093] a. Prepare feeder layer cells for passage one day in advance. Treat 4-well plates with 0.1% gelatin aqueous solution, incubate at 37°C for 30 min, and then discard. Add 6×10 cells to the 4-well plates. 5 The culture plate was placed in a 37°C incubator for more than 6 h.

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

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

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

[0097] First, the morphology of the obtained gESCs was observed, and it was found that the gESCs cells had a small diameter, a large nucleus, a prominent nucleolus, less cytoplasm, a larger nucleoplasm, and a dome-shaped growth. The cell clones had clear edges, a smooth surface, and refractive index, which was consistent with the initial gESCs morphology. After multiple passages, the morphology did not change significantly. Figure 2 A.

[0098] When the confluence of goat embryonic stem cells reached 70%, they were fixed with 4% paraformaldehyde for 1 min, and the alkaline phosphatase staining kit was strictly followed. The cells were incubated at room temperature in the dark for 15 min, and then examined under a microscope. Figure 2 B, AP staining results showed that gESCs were positive for alkaline phosphatase.

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

[0100] Goat fetal fibroblasts and goat embryonic stem cells were inoculated into 12-well plates, with 3 replicates each. After 3 days of culture, the cells were collected and tested using a cell cycle kit. The specific operation was carried out according to the steps in the kit manual. The analysis results are shown in Figure 3 The results showed that compared with goat fibroblasts, the number of cells in the G1 and S phases of the goat embryonic stem cell line was significantly higher than that of goat fetal fibroblasts, P<0.01.

[0101] 3. Analysis of expression of pluripotency marker genes in goat embryonic stem cells

[0102] Total RNA from goat embryonic stem cells (gESCs) cultured under 4i / LA conditions and goat fetal fibroblasts (GFF) cultured under fibroblast culture conditions were extracted and hydrolyzed with nuclease-free water. After measuring the concentration and purity of RNA, reverse transcription was used to synthesize cDNA library, and the expression of OCT4, SOX2, NANOG, KLF4, DPPA3, SALL4, and REX1 was analyzed. The results are shown in Figure 4 As shown in the figure, 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 homeodomain-containing transcription factor that is required for the maintenance of 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 the confluence of gESCs reached about 80%, the cells were fixed with 4% PFA for 10 min, and then immunofluorescence staining was performed to detect the expression of pluripotent genes OCT4, SOX2, and NANOG. As shown in the results, OCT4, SOX2, and NANOG proteins were expressed normally (see Figure 5 ), cell surface markers SSEA4, TRA-1-60 and TRA-1-81 were expressed normally (see Figure 6 ).

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

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

[0107] Karyotype analysis of gESCs was performed to identify whether there was any chromosomal aberration. The results showed that the number of chromosomes in the cells was normal, 2n = 60 (see Figure 7 A), which can be used for subsequent experiments.

[0108] gESCs were digested into single cells and seeded at a certain density in a U-shaped culture dish with low adsorption density. The culture was suspended for 7 days and the medium was changed every other day. After 7 days, the formed embryoid bodies were transferred to gelatin-coated cell culture plates and cultured for 28 days. The results are as follows Figure 7As shown in B, the cells can form embryoid bodies with spherical structures. Immunofluorescence staining was performed on the cells differentiated in vitro. In the figure, β-Tubulin is a cytoskeletal protein and 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 a variety of nervous system tumor cells, which is a neuroectoderm marker; AFP is an endoderm marker. The results showed that the mesoderm marker proteins β-Tubulin and α-SMA, the ectoderm marker protein Nestin, and the endoderm marker protein AFP were all expressed normally (see Figure 7 ). Subsequently, RT-qPCR was performed to detect the marker genes of the three germ layers, and the results showed that the marker genes of each germ layer were significantly expressed (see Figure 8 )(P<0.05). This indicates that 4i / LA-gESCs cultured in the 4i / LA system proposed in the present invention have the potential to differentiate into the three germ layers in vitro.

[0109] 5. Differentiated teratoma in vivo

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

[0111] The Chinese names of the English nouns involved in the present invention are listed in Table 1.

[0112] Table 1 Chinese and English noun comparison table

[0113] Serial number Chinese name English name 1 Dulbecco's Phosphate Buffered Saline D-PBS 2 Activin A ActivinA 3 Goat Embryonic Stem Cells gESCs 4 Alkaline phosphatase AP 5 Paraformaldehyde PFA 6 Goat fetal fibroblasts GFF 7 Nuclease-free water RNase-free water name Brand and specifications 1 Acidic benchtop fluid Sigma, T1788-100mL 2 Alkaline Phosphatase Staining Kit Blue Sky, P0321S

[0114] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0115] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A goat embryonic stem cell culture system, characterized in that: The goat embryonic stem cell culture system is based on mTeSR TM Plus is the basic culture medium, and is supplemented with: 20ng / mL~25ng / mL of leukemia inhibitory factor LIF, 1μM~1.5μM of PD0325901, 4μM~5μM of IWR-1, 45μg / mL~50μg / mL of vitamin C, 15ng / mL~20ng / mL of activin A, 1~1.2μM of CGP77675, and 1.5~2μM of GO6983.

2. The goat embryonic stem cell culture system according to claim 1, characterized in that: The goat embryonic stem cell culture system is based on mTeSR TM Plus is the basic culture medium, and is supplemented with: 25 ng / mL leukemia inhibitory factor, 1 μM PD0325901, 5 μM IWR-1, 50 μg / mL vitamin C, 20 ng / mL activin A, 1.2 μM CGP77675, and 2 μM GO6983.

3. A method for establishing a goat embryonic stem cell line, characterized in that: The steps include: Preparation of feeder layer cells: Mouse fetal fibroblasts were isolated and subcultured, treated with mouse fetal fibroblast culture medium containing 10 μg / mL to 15 μg / mL mitomycin C for 3 h to 4 h, and then digested to obtain mouse fetal fibroblasts as feeder layer cells; Whole embryo inoculation method to isolate goat embryonic stem cells: using the culture system described in claim 1 to isolate goat embryonic stem cells, and then inoculating the isolated goat embryonic stem cells into feeder layer cells at a ratio of 1:1 to 3 to obtain goat embryonic stem cells; Subculturing the obtained goat embryonic stem cells to obtain a goat embryonic stem cell line; The formula of 50mL of the mouse fetal fibroblast culture medium containing 10μg / mL to 15μg / mL mitomycin C is: 45mL DMEM high-glucose medium+5mL fetal bovine serum+500μL penicillin-streptomycin double antibody+10μg / mL to 15μg / mL mitomycin C.

4. The method for establishing goat embryonic stem cell line according to claim 3, characterized in that: The mouse fetal fibroblast culture medium containing 10 μg / mL mitomycin C was used to treat mouse fetal fibroblasts during feeder cell preparation.

5. The method for establishing goat embryonic stem cell line according to claim 3, characterized in that: Feeder cell preparation includes the following steps: Mouse fetal fibroblasts were cultured in mouse fetal fibroblast culture medium until the cell confluence reached more than 75%, and then the cells were passaged at a ratio of 1:4-5; After the subculture, when the mouse fetal fibroblasts completely cover the surface of the culture dish again, the mouse fetal fibroblast culture medium is replaced with a culture medium containing 10 μg / mL to 15 μg / mL mitomycin C and the cells are treated for 3 hours; The mouse fetal fibroblast culture medium containing 10 μg / mL to 15 μg / mL mitomycin C was discarded, the mouse fetal fibroblasts were rinsed with D-PBS, and then the mouse fetal fibroblasts were digested with 0.25% trypsin to obtain a mouse fetal fibroblast suspension; The diluted mouse fetal fibroblast cell suspension containing DMSO was 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.

6. The method for establishing goat embryonic stem cell line according to claim 3, characterized in that: The process of isolating goat embryonic stem cells by whole embryo inoculation is as follows: Goat blastocysts with good morphology and obvious inner cell mass are selected for in vitro isolation of goat embryonic stem cells, the zona pellucida of the blastocyst is removed using an acidic benchtop solution, and the culture system described in claim 1 is used to culture and isolate goat embryonic stem cells.

7. The method for establishing goat embryonic stem cell line according to claim 3, characterized in that: The subculture process of goat embryonic stem cells is as follows: Remove the original culture medium of goat embryonic stem cells and add D-PBS to gently shake and rinse 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, and discard the supernatant after centrifugation; Resuspend the cells in goat embryonic stem cell culture medium containing Y-27632 at a final concentration of 10 μM to 15 μM, and passage them at a ratio of 1:3 to 10 according to the cell growth density; During subculturing, the goat embryonic stem cell culture medium without Y-27632 was replaced every 24 hours, and the next subculturing could be performed when the cell confluence reached 80% to 90%.

8. A method for constructing a teratoma animal model, characterized in that: The goat embryonic stem cells were cultured using the method of claim 3, and 1x10 6 ~2x10 6 Goat embryonic stem cells with a cell number were resuspended in 200 μL to 300 μL of matrix gel and injected subcutaneously into mice. After 9-10 weeks, teratomas were formed.

9. The method for constructing a teratoma animal model according to claim 8, characterized in that: The teratoma has ectoderm, mesoderm and endoderm.

10. Use of a teratoma animal model in screening drugs for treating teratoma, characterized in that: The teratoma animal model is constructed by the method of claim 8.

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