Goose primordial germ cell separation medium and application thereof
By optimizing the culture medium and conditions for isolating goose primordial germ cells, the problems of severe cell differentiation and slow proliferation in the goose PGC culture system were solved, achieving efficient and stable cell isolation and gene editing applications, and improving the efficiency of goose breeding and gene editing.
Patent Information
- Application Number
- CN202510915377.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-03
- Publication Date
- 2025-11-14
AI Technical Summary
Existing goose primordial germ cell culture systems suffer from severe cell differentiation, slow proliferation rates, and difficulty in long-term maintenance, making it difficult to meet the efficiency and stability requirements for application and becoming a bottleneck restricting technological breakthroughs in the goose field.
A specific formula of goose primordial germ cell isolation medium, containing 18-22 mL of B-27 supplement, 1-3 mM of pro-glutamyl dipeptide, and other components in calcium-free DMEM medium, combined with goose-derived and human-derived growth factors, was used for the isolation and culture of whole blood from goose embryos. The optimized culture conditions were 37-39℃ and 5% CO2.
It achieves stable and efficient isolation and long-term maintenance of goose primordial germ cells, which still have good chimeric goose embryo production performance after cryopreservation. The survival rate of surrogate embryos after 16 days can reach up to 57%, and the cell performance after gene editing is maintained. It is suitable for breeding, hybridization breeding and gene editing.
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Figure CN120944807A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a culture medium for isolating goose primordial germ cells and its application. Background Technology
[0002] Primordial germ cells (PGCs) are a group of undifferentiated progenitor cells present in early embryos, playing a crucial role in developing into functional gametes and transmitting parental genetic material to offspring. In avian development, PGCs originate from germplasm deposited in the mother and actively enter the circulatory system in the very early stages of embryonic development. They migrate with the bloodstream to the lateral plate region of the embryo and eventually settle in the genital ridge, where they proliferate and differentiate to form a population of germ cells within the gonads. PGCs can be obtained by extracting blood from early embryos, amplified through in vitro culture or genetically modified, and then microinjected back into the blood vessels of the recipient embryo. These PGCs can successfully home to the recipient gonads and participate in gamete formation, ultimately producing offspring carrying the donor's genetic information. This technological approach provides a powerful biotechnological platform for avian genetic resource preservation, gene editing, breeding of superior breeds, and hybridization.
[0003] Currently, the isolation of avian progenitor cells (PGCs) mainly relies on their early vascular migration characteristics. The key technology involves extracting trace amounts of blood from embryos during specific developmental windows using precise micromanipulation, and then enriching and obtaining PGCs from this blood. Chickens, as a model organism, have seen significant breakthroughs in PGC in vitro culture technology. Key advancements include the development of serum-free, low-calcium culture media with defined components, replacing the complex culture systems that previously relied on fetal bovine serum or feeder cells, greatly simplifying the process and improving reproducibility. Simultaneously, PGC transplantation technology has been optimized, such as using intraovarian injection via eggshell fenestration instead of traditional extraovarian injection, and continuing to culture the injected embryos within surrogate eggshells until hatching, significantly improving recipient embryo survival rates and the rate of obtaining healthy chicks. The maturity of these technologies has strongly promoted the research and application of chicken gene editing and germplasm cryopreservation.
[0004] PGC-based biotechnology has profound significance for poultry genetic improvement and germplasm resource conservation. It offers a potential solution, particularly to the severe challenges facing the goose industry. Current germplasm conservation mainly relies on live animal preservation, which suffers from high costs, easy loss of genetic diversity, and high disease risk, severely restricting the efficiency of goose breeding and propagation. However, establishing an efficient and stable in vitro culture system for PGCs suitable for geese presents significant challenges. The core difficulty lies in the significant differences in the molecular regulatory mechanisms of different poultry PGCs. For example, the long-term self-renewal and proliferation of chicken PGCs in vitro clearly depends on the synergistic effects of signaling pathways such as insulin, fibroblast growth factor 2 (FGF2), and activin. However, duck-derived PGCs can only maintain limited proliferation briefly in simulated chicken culture conditions (FAOcs medium containing FGF2, Activin, oocyte transferrin, and chicken serum), failing to reach the required order of magnitude (>10,000) for application. The proliferation of progenitor cells (PGCs) in the zebra finch is highly dependent on bone morphogenetic protein 4 (BMP4), and its effect only lasts for about two weeks. The fundamental differences in the response patterns to core growth factors, coupled with a lack of systematic understanding of the developmental regulatory network of goose PGCs, have led to widespread problems in currently reported goose PGC culture systems, including severe cell differentiation, slow proliferation rates, and difficulty in long-term maintenance. These existing systems fall far short of the efficiency and stability requirements for in vivo manipulation, becoming a bottleneck restricting the breakthrough of this technology in the goose field. Summary of the Invention
[0005] Purpose of the invention: The purpose of this invention is to provide a culture medium suitable for isolating goose primordial germ cells; the second purpose is to provide goose primordial germ cells prepared using this culture medium and their applications.
[0006] Technical Solution: The goose primordial germ cell isolation culture medium of the present invention contains 18-22 mL B-27 supplement, 1-3 mM pro-glutamyl dipeptide, 8-12 mL MEM non-essential amino acid solution, 5-15 mL nucleoside, 0.08-0.16 mM β-mercaptoethanol, 1-4 g ovalbumin, 0.8-1.6 mM pyruvate, 0.1-0.3 mM calcium chloride, 0.1-0.2 g heparin sodium, 40-60 μg insulin-like growth factor 1, 6-10 μg fibroblast growth factor 2, 23-27 μg bone morphogenetic protein 4, 23-27 μg stem cell factor, 23-27 μg leukemia inhibitory factor per liter, and the remainder is calcium-free DMEM culture medium with a glucose concentration of 10-14 mM.
[0007] Preferably, both insulin-like growth factor 1 and fibroblast growth factor 2 are human-derived proteins, wherein fibroblast growth factor 2 is a thermostable mutant with the amino acid sequence shown in SEQ ID NO: 1;
[0008] The bone morphogenetic protein 4, stem cell factor, and leukemia inhibitory factor are all goose-derived proteins, wherein bone morphogenetic protein 4 has the amino acid sequence shown in SEQ ID NO: 2, stem cell factor has the amino acid sequence shown in SEQ ID NO: 3, and leukemia inhibitory factor has the amino acid sequence shown in SEQ ID NO: 4.
[0009] The goose primordial germ cells prepared using the aforementioned goose primordial germ cell isolation culture medium, as described in this invention, are obtained through the following steps:
[0010] (1) After the goose eggs have been incubated for 60-84 hours, 2-4 μL of whole blood is extracted from the goose embryos;
[0011] (2) The obtained whole blood was mixed with 60-70 times the volume of goose primordial germ cell isolation culture medium and then cultured.
[0012] (3) Change the culture medium every 1-3 days to continuously culture and obtain goose primordial germ cells.
[0013] Preferably, in the separation step 1, the goose eggs are from any one of the following goose species: Huoyan goose, Wuzong goose, Huoyan goose, Guangfeng goose, Lionhead goose, Wanxi white goose, Sichuan white goose, and Magang goose.
[0014] Preferably, in the separation step 1, whole blood is drawn from the dorsal artery of the goose embryo.
[0015] Preferably, in the separation step, the culture environment is 37-39℃ and 5% CO2.
[0016] The application of the goose primordial germ cells described in this invention in goose conservation and rebreeding.
[0017] The application of the goose primordial germ cells described in this invention in goose hybridization breeding.
[0018] The application of the goose primordial germ cells described in this invention in the propagation of superior goose breeds.
[0019] The application of goose primordial germ cells described in this invention in goose gene editing.
[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The culture medium of the present invention can effectively avoid the problems of low quantity, high differentiation, and difficulty in long-term maintenance when preparing goose primordial germ cells in the existing system, and is more stable and efficient; 2. Primordial germ cells prepared using this culture medium still have good chimeric goose embryo production performance after cryopreservation, and the survival rate of surrogate embryos after 16 days can reach up to 57%. At the same time, chimeric geese have normal reproductive capacity and can be used for breeding and hybridization; 3. Primordial germ cells prepared using this culture medium still retain the same level of cell performance after gene editing and can be used for gene-edited goose production. Attached Figure Description
[0021] Figure 1 The images show the observation results of PGCs cultured on Huoyan goose for 20 days. The left image shows PGCs isolated from the control culture medium, and the right image shows PGCs isolated from the goose primordial germ cell isolation culture medium.
[0022] Figure 2 Figure showing the identification results of PGCs from goose primordial germ cells isolated using the goose primordial germ cell isolation culture medium;
[0023] Figure 3 A schematic diagram of the hybrid offspring produced by the crossbreeding of Lionhead Goose and Hollow-Eyed Goose;
[0024] Figure 4 Figure 1 shows the observation results of Taizhou goose PGCs cells expressing red fluorescence after gene editing.
[0025] Figure 5 A schematic diagram of the hybrid offspring produced by gene editing and chimerism of Taizhou geese. Detailed Implementation
[0026] The technical solution of the present invention will be further described below.
[0027] Example 1: Stable and efficient in vitro culture and identification of goose primordial germ cells (PGCs)
[0028] 1. Preparation of culture medium for isolating goose primordial germ cells
[0029] (1) Based on the amino acid sequence of human thermostable fibroblast growth factor 2 as shown in SEQ ID NO: 1, the amino acid sequence of goose bone morphogenetic protein 4 as shown in SEQ ID NO: 2, the amino acid sequence of goose stem cell factor as shown in SEQ ID NO: 3, and the amino acid sequence of goose leukemia inhibitory factor as shown in SEQ ID NO: 4, the nucleotide sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. after codon optimization, cloned into the pUC57 vector, and expressed using the HEK293 cell system to obtain the above four proteins;
[0030] (2) Take 20 mL of B-27 supplement (Gibco, catalog number 17504001), 10 mL of GlutaMAX supplement (Gibco, catalog number 35050061), 10 mL of MEM non-essential amino acid solution (Gibco, catalog number 11140050), 10 mL of EmbryoMax nucleoside (Sigma-Aldrich, catalog number ES008), 0.12 mmol β-mercaptoethanol, 2 g ovalbumin (Sigma-Aldrich, A5530), 1.2 mmol pyruvate, 0.2 mmol calcium chloride, 0.15 g heparin sodium (MCE, catalog number HY-17567A), and 50 μg human insulin-like growth factor 1 (R&D). Systems (catalog number 291-G1), and the aforementioned 8 μg human heat-stable fibroblast growth factor 2, 25 μg goose bone morphogenetic protein 4, 25 μg goose stem cell factor, and 25 μg goose leukemia inhibitory factor, were used to dilute calcium-free high-glucose DMEM medium with calcium-free low-glucose DMEM medium to obtain DMEM medium with a final glucose concentration of 12 mM. The aforementioned components were then added to 1 L of this medium to obtain goose primordial germ cell isolation medium.
[0031] (3) Take 20 mL of B-27 supplement (Gibco, catalog number 17504001), 10 mL of GlutaMAX supplement (Gibco, catalog number 35050061), 10 mL of MEM non-essential amino acid solution (Gibco, catalog number 11140050), 10 mL of EmbryoMax nucleoside (Sigma-Aldrich, catalog number ES008), 0.12 mmol β-mercaptoethanol, 2 g ovalbumin (Sigma-Aldrich, catalog number A5530), 1.2 mmol pyruvate, 0.2 mmol calcium chloride, 0.15 g heparin sodium (MCE, catalog number HY-17567A), and 50 μg human insulin-like growth factor 1 (R&D). Systems, catalog number 291-G1), 8 μg human fibroblast growth factor 1 (R&D Biosystems, catalog number 232-FA-025 / CF), 8 μg human fibroblast growth factor 2 (R&D Biosystems, catalog number 3718-FB-010), 25 μg human bone morphogenetic protein 4 (PeproTech, catalog number 120-05ET-01M). Calcium-free high-glucose DMEM medium was diluted with calcium-free low-glucose DMEM medium to obtain DMEM medium with a final glucose concentration of 12 mM. The aforementioned components were then added to 1 L of this medium to obtain the control medium.
[0032] 2. Isolation and culture of PGCs from the 'Hooyan Goose'
[0033] (1) Take 10 peep-eyed goose eggs and incubate them in an incubator at 38℃ and 70% humidity for 72 hours;
[0034] (2) Make a 0.5 cm diameter window on the eggshell, rotate the goose egg until the goose embryo is exactly at the window, and under a stereomicroscope, use a sterile pipette with a capillary glass needle attached to the end to draw 3 μL of whole blood from the dorsal artery of the goose embryo, add 0.2 mL of the goose primordial germ cell isolation culture medium or control culture medium prepared above, and culture at 38°C and 5% CO2.
[0035] (2) Every 2 days, discard one-third of the culture medium and add the same volume of fresh goose primordial germ cell isolation medium or control medium.
[0036] (3) Morphological observation and identification were performed after 20 days of culture;
[0037] (4) Collect the cell culture medium separated from the goose primordial germ cell separation medium, centrifuge at 2000 rpm for 4 min, discard the supernatant, resuspend the cell pellet in 200 μL of 4% paraformaldehyde (PFA) fixative, and fix at room temperature for 20 min.
[0038] Centrifuge again at 2000 rpm for 4 min, discard the supernatant and wash the cells twice with 300 μL PBS;
[0039] Add 200 μL of 0.5% Triton X 100-PBS solution, permeabilize at room temperature for 15 min, centrifuge at 2000 rpm for 4 min, discard the supernatant, and block the cells with 0.5% sheep serum-PBS solution for 30 min;
[0040] Add 250-fold diluted mouse anti-human SSEA-1 and rabbit anti-human DAZL antibodies, incubate overnight at 4°C, centrifuge at 2000 rpm for 4 min, discard the supernatant and wash the cells three times with 300 μL PBS;
[0041] Add 500-fold diluted donkey anti-mouse IgM-AF488 and donkey anti-rabbit IgG-AF568 antibodies, incubate for 1 h, centrifuge at 2000 rpm for 4 min, discard the supernatant and wash the cells three times with 300 μL PBS;
[0042] Cell nuclei were stained using Hoechst staining. After incubation for 15 min, the cells were centrifuged at 2000 rpm for 4 min, the supernatant was discarded, and the cells were washed three times with 300 μL PBS. The cells were then resuspended in 10 μL PBS, added to a glass slide, and mounted. The cells were then identified using a fluorescence microscope.
[0043] Morphological observation of isolated PGCs as follows Figure 1As shown, although the PGCs of Huoyan goose isolated from the control culture medium could grow, their number was small and there were many spindle cells, indicating that they were more differentiated; while the PGCs of Huoyan goose isolated from the goose primordial germ cell isolation culture medium were more numerous, had better morphology, and no differentiated cells could be observed.
[0044] The identification results of isolated PGCs are as follows: Figure 2 As shown, the PGCs isolated from goose primordial germ cells in the goose primordial germ cell isolation medium were positive for both SSEA-1 and DAZL, indicating that they possess PGC characteristics.
[0045] 3. Isolation and culture of PGCs from different local goose breeds
[0046] Using the aforementioned method, pGCs from Wuzong goose, Huoyan goose, Guangfeng goose, Lionhead goose, Wanxi white goose, Sichuan white goose, and Magang goose were isolated and cultured for a long period using goose primordial germ cell isolation medium. The results are shown in Table 1.
[0047] Table 1. Isolation and culture results of PGCs from different local goose breeds
[0048] variety Number of days of cultivation (days) Cell line establishment success rate Black-haired goose 63 9 / 18(50％) Goose with a wide eye 63 5 / 10(50％) Guangfeng Goose 60 3 / 9(33％) Lionhead Goose 60 4 / 14(29％) Anhui White Goose 52 4 / 17(24％) Sichuan White Goose 52 4 / 14(29％) Magang Goose 52 6 / 13(46％)
[0049] The results showed that PGCs isolated using goose primordial germ cell isolation medium could maintain their function for a long time, and the success rate of cell line establishment could reach 50%, making it a stable and efficient method for isolating goose PGCs.
[0050] Example 2: Application of goose PGCs technology to the cryopreservation, rebreeding, and hybridization of Lionhead geese
[0051] 1. Cryopreservation, thawing, and large-scale reculture
[0052] (1) Lionhead goose PGCs isolated in Example 1 were cultured in 24-well plates. When the total number of cells per well exceeded 1×10⁻⁶, the culture was considered complete. 6 At that time, Stem Cellbanker cryopreservation solution (ZENOAQ, catalog number 11924) was used at 2 × 10⁻⁶. 5 Cells / tubes were cryopreserved, and the cells were cooled to -80°C at a rate of 1°C per minute using a programmed cooling box, and then transferred to liquid nitrogen for long-term storage.
[0053] (2) The Lionhead Goose PGCs that have been frozen in liquid nitrogen for 6 months were thawed in a water bath at 37°C for 1 minute, centrifuged at 2000 rpm for 4 minutes, the cryopreservation solution was removed, the cells were resuspended in goose PGCs-specific culture medium, the cell suspension was transferred to a 48-well cell culture plate and cultured at 38°C and 5% CO2.
[0054] (3) Every 2 days, discard one-third of the culture medium and add the same volume of fresh goose primordial germ cell isolation medium or control medium.
[0055] (4) When the cells reach a confluence of 50%, take half of the cells for passage and expand the cell culture until the total number of cells reaches 1×10⁶. 5 Collect cells at a rate of 1 cell / well or higher for subsequent experiments;
[0056] 2. Production of chimeric goose embryos via surrogacy
[0057] (1) Using the high-egg-producing geese as surrogate geese, the fertilized goose eggs were incubated in an incubator at 38°C and 70% humidity for 72 hours. Then, a window with a diameter of 0.5 cm was opened on the eggshell. The goose egg was rotated until the goose embryo was exactly at the window. Under a stereomicroscope, a sterile pipette with a capillary glass needle attached to the end was used to inject the PGCs cell suspension obtained above after recovery and expansion culture with 0.5% fastgreen dye (Sigma-Aldrich, catalog number: 2353-45-9) as an injection indicator into the dorsal artery of the goose embryo.
[0058] (2) After the injection, add 50 μL of penicillin-streptomycin antibiotic (Gbico, catalog number: 15140122) through the window on the goose eggshell and seal the window on the shell with medical tape with cotton cloth matrix.
[0059] (3) The chimeric embryos that have been injected are placed in an incubator at 38°C and 70% humidity to continue incubation until hatching, thereby obtaining surrogate chimeric geese.
[0060] The number of PGCs injected and the results are shown in Table 2. The survival rate of 16-day chimeric gosling surrogate embryos from Lionhead Goose A-derived PGCs can reach 33%, and the survival rate of 16-day chimeric gosling surrogate embryos from Lionhead Goose B-derived PGCs can reach 53%.
[0061] Table 2. Number and results of PGCs cell injection
[0062]
[0063] 3. Producing offspring of surrogate chimeric geese through rebreeding
[0064] (1) After hatching, the male and female geese of the surrogate chimeric goose are raised together until the female goose begins to lay eggs. The eggs are collected and incubated in an incubator at 38°C and 70% humidity.
[0065] (2) Determine the specific offspring type of the goslings by phenotypic analysis.
[0066] The results are as follows Figure 3As shown, the gray goslings are Lionhead geese, and the yellow goslings are Hawthorn geese, indicating that chimeric geese produced from goose PGCs isolated by goose primordial germ cell isolation culture medium after cryopreservation and thawing can successfully produce offspring for crossbreeding.
[0067] 4. Hybrid breeding of surrogate chimeric geese
[0068] By crossbreeding the surrogate chimeric geese obtained above with normal open-eyed geese, the hybridization of Lionhead geese and open-eyed geese can be achieved.
[0069] The results are shown in Table 3. The surrogate chimeric geese and normal open-eyed geese can be successfully hybridized and produce offspring with the Lion Head Goose trait.
[0070] Table 3 Results of hybridization breeding of surrogate chimeric geese
[0071]
[0072] Example 3: Application of goose PGCs technology in the production of red fluorescent gene-edited geese
[0073] 1. Establishment of td Tomato fluorescent transgenic PGCs cell lines
[0074] (1) Taizhou goose PGCs cell line was isolated and cultured according to the method described in Example 1;
[0075] (2) Insert the td Tomato fluorescent gene as shown in SEQ ID NO: 5 into the piggybac plasmid (Addgene, catalog number 203312) to obtain the piggybac plasmid carrying the transposon td Tomato fluorescent gene; use the Lipofectamin2000 kit to prepare a transfection mixture containing 2.5 μg of the piggybac plasmid carrying the transposon td Tomato fluorescent gene and 2.5 μg of the helper plasmid carrying the transposase (U-Bio Biotechnology, catalog number: VT1664);
[0076] (3) Resuspend 2×10⁻⁶ cells in the transfection mixture. 5 Taizhou goose PGCs cells were incubated at 38°C and 5% CO2 for 6 hours.
[0077] (4) After incubation, centrifuge to remove the transfection mixture, suspend the cells in goose PGCs culture medium, and continue to culture at 38°C and 5% CO2.
[0078] (5) Six days after transfection, puromycin was added to the cell culture medium at a final concentration of 20 ng / mL until all cells that did not express td Tomato fluorescent protein died, thus obtaining a stable Taizhou goose PGC cell line expressing td Tomato fluorescence.
[0079] The results are as follows Figure 4 As shown, all edited goose PGCs cells expressed red fluorescence.
[0080] 2. Production of red fluorescent gene-edited geese
[0081] (1) Taizhou geese were selected as surrogate geese. Their fertilized goose eggs were incubated in an incubator at 38°C and 70% humidity for 72 hours. Then, a window with a diameter of 0.5 cm was opened on the eggshell. The goose egg was rotated until the goose embryo was exactly at the window. Under a stereomicroscope, a sterile pipette with a capillary glass needle attached to the end was used to inject the Taizhou goose PGCs that stably expressed td Tomato fluorescence obtained above into the dorsal artery of the goose embryo with 0.5% fastgreen dye as an injection indicator.
[0082] (2) After the injection, add 50 μL of penicillin-streptomycin antibiotic through the window on the goose eggshell and seal the window on the shell with medical tape with cotton cloth substrate.
[0083] (3) The chimeric embryos that have been injected are placed in an incubator at 38°C and 70% humidity to continue incubation until hatching, thereby obtaining surrogate chimeric goslings.
[0084] The number of PGCs injected and the results are shown in Table 4. The survival rate of surrogate embryos after 16 days can reach 57%.
[0085] Table 4. Number and results of PGCs cell injection in surrogate chimeric geese
[0086]
[0087] (4) After the chimera geese hatch, the male and female geese are raised together until the female geese start laying eggs. The eggs are then collected and incubated in an incubator at 38°C and 70% humidity.
[0088] (2) Determine the specific offspring type of the goslings by phenotypic analysis.
[0089] The results are as follows Figure 5 As shown in Table 5, among which Figure 5 The second gosling from the left is a gene-edited Taizhou goose expressing red fluorescence, indicating that chimeric geese produced after gene editing using goose primordial germ cell isolation culture medium can successfully produce offspring.
[0090] Table 5 Results of hybridization breeding of gene-edited chimeric geese
[0091]
Claims
1. A culture medium for isolating goose primordial germ cells, characterized in that, Each liter of the culture medium contains 18-22 mL of B-27 supplement, 1-3 mM pro-glutamyl dipeptide, 8-12 mL of MEM non-essential amino acid solution, 5-15 mL of nucleoside, 0.08-0.16 mM β-mercaptoethanol, 1-4 g of ovalbumin, 0.8-1.6 mM pyruvate, 0.1-0.3 mM calcium chloride, 0.1-0.2 g of heparin sodium, 40-60 μg of insulin-like growth factor 1, 6-10 μg of fibroblast growth factor 2, 23-27 μg of bone morphogenetic protein 4, 23-27 μg of stem cell factor, 23-27 μg of leukemia inhibitory factor, and the remainder is calcium-free DMEM medium containing 10-14 mM glucose.
2. The goose primordial germ cell isolation culture medium according to claim 1, characterized in that, The insulin-like growth factor 1 and fibroblast growth factor 2 are human-derived proteins, wherein fibroblast growth factor 2 is a thermostable mutant with the amino acid sequence shown in SEQ ID NO: 1; The bone morphogenetic protein 4, stem cell factor, and leukemia inhibitory factor are goose-derived proteins, wherein bone morphogenetic protein 4 has the amino acid sequence shown in SEQ ID NO: 2, stem cell factor has the amino acid sequence shown in SEQ ID NO: 3, and leukemia inhibitory factor has the amino acid sequence shown in SEQ ID NO:
4.
3. A goose primordial germ cell prepared using the goose primordial germ cell isolation culture medium according to claim 1, characterized in that, The cells were obtained by the following steps: (1) After the goose eggs have been incubated for 60-84 hours, 2-4 μL of whole blood is extracted from the goose embryos; (2) The obtained whole blood was mixed with 60-70 times the volume of goose primordial germ cell isolation culture medium and then cultured. (3) Change the culture medium every 1-3 days to continuously culture and obtain goose primordial germ cells.
4. The goose primordial germ cell according to claim 3, characterized in that, In separation step 1, the goose eggs are from any one of the following goose species: Huoyan goose, Wuzong goose, Huoyan goose, Guangfeng goose, Lionhead goose, Wanxi white goose, Sichuan white goose, and Magang goose.
5. The goose primordial germ cell according to claim 3, characterized in that, In separation step 1, whole blood is drawn from the dorsal artery of the goose embryo.
6. The goose primordial germ cell according to claim 3, characterized in that, In the separation step, the culture environment is 37-39℃ and 5% CO2.
7. The application of the goose primordial germ cell as described in claim 3 in goose conservation and rebreeding.
8. The application of the goose primordial germ cell as described in claim 3 in goose hybridization breeding.
9. The application of the goose primordial germ cell as described in claim 3 in the propagation of improved goose breeds.
10. The application of the goose primordial germ cell as described in claim 3 in goose gene editing.