A method for establishing a line of primordial germ cells derived from chicken gonads

Abstract

The application provides a method for establishing a line of primordial germ cells from a chicken gonad, which comprises the steps of aseptically collecting chicken embryo gonad tissue from a 28-30HH period fertilized chicken egg, separating PGCs, in-vitro culturing PGCs and establishing a line; the scheme of the application realizes the separation of PGCs from gonad tissue blocks by a specific aperture screen separation device and a specific separation medium, and realizes in-situ purification while collecting PGCs; and then stable PGC clones are obtained through in-vitro culturing; on the one hand, the application discards the digestion step that cannot be bypassed in the prior art, and avoids the damage to key functional proteins on the cell membrane surface; on the other hand, the subsequent centrifugation, complex sorting and other operation steps are significantly reduced, the secondary damage to cells caused by physical stress is minimized, and the cell survival rate and initial activity are improved; the method of the application only needs conventional cell culture equipment, is simple to operate and has good repeatability.

Description

Technical Field

[0001] This invention belongs to the fields of animal husbandry and biotechnology, and relates to a method for obtaining chicken primordial germ cells from the gonads of chicken embryos, specifically a method for establishing a lineage of primordial germ cells derived from chicken gonads. Background Technology

[0002] The precursors of avian reproductive cells (sperm and egg) are primordial germ cells (PGCs), which can differentiate into reproductive cells during development. They have pluripotency and self-renewal capabilities and have important applications in avian gene editing, transgenic poultry production, drug screening, and poultry breeding.

[0003] For chickens, due to the unique structure of their reproductive system, it is impossible to achieve "ex-situ preservation" through the freezing of gametes commonly used in mammals. Therefore, the cryopreservation of PGCs has become the only effective means of protecting chicken germplasm resources. At the same time, chicken gene editing technology mainly relies on modifying the genes of PGCs in vitro, then injecting them into recipient embryos through microinjection to obtain chimeras, and then selecting and mating to obtain homozygous gene-edited individuals. Therefore, efficiently obtaining highly active chicken PGCs is a prerequisite for the application of the above technologies.

[0004] Currently, there are two main ways to obtain chicken progenitor cells (PGCs): one is to isolate them from the blood of early embryos (14-17HH stage), and the other is to isolate them from the gonads of early embryos (e.g., 28-30HH stage). Gonad-derived PGCs have the following advantages: compared to blood-derived PGCs, gonad-derived PGCs have already completed their migration to the genital ridge, their cell differentiation is closer to that of germ cell precursors, and they have a stronger potential for in vitro directed differentiation into gametes; furthermore, the content of PGCs in gonadal tissue is relatively stable and less affected by contaminating cells in the blood.

[0005] Currently, there are four main techniques for isolating PGCs from chicken embryo gonads: 1) mechanically cutting and digesting the gonads; 2) using differential adhesion properties for separation; 3) using Ficoll gradient centrifugation; and 4) flow cytometry sorting (FACS).

[0006] The first method, "mechanically dissecting the gonads followed by digestion," involves mechanically dissecting the gonads and then digesting them with digestive enzymes (such as trypsin or collagenase). This method can yield a large number of cells, but the surface properties of PGCs may be affected, thus influencing their adhesion behavior. Methods 2) to 4) also require first mechanically dissecting the gonads and then digesting them with digestive enzymes. PGCs are then separated by utilizing the difference in adhesion characteristics between PGCs and other cells (such as fibroblasts) on the culture dish (method 2), or by using Ficoll gradient centrifugation (method 3), or by using fluorescent labeling and flow cytometry sorting techniques (method 4).

[0007] The four existing technical methods described above have the following two technical defects: On the one hand, to obtain sufficient cell quantity and separation efficiency, the four existing technical methods mentioned above all require "mechanical shearing" and "digestion" operations, which inevitably cut or damage key functional proteins (such as glycoproteins, adhesion molecules, and homing receptors) on the surface of PGC cell membranes. Although this damage does not affect the short-term cell viability assessment, it severely weakens the core ability of PGCs to subsequently hom to the receptor gonad and develop into functional gametes in vivo, resulting in impaired function of the separated PGCs and greatly limiting their practical effectiveness in downstream applications requiring cell transplantation (such as chimera preparation).

[0008] On the other hand, in order to obtain high-purity PGCs after digestion, existing technologies require purification steps such as multiple centrifugation and washing, complex gradient centrifugation, or expensive flow cytometry cell sorting. These subsequent operations are cumbersome, and repeated centrifugation and other physical stresses can cause secondary damage to cells, further reducing cell viability and increasing the risk of contamination.

[0009] Therefore, the functional integrity of PGCs obtained by current technologies is insufficient; in particular, there is insufficient support for female PGCs, the establishment time for female PGCs is significantly longer than that for males, and the success rate is low. This has become a thorny technical difficulty in avian reproductive technology, especially in the field of oocyte development and genetic resource preservation. Summary of the Invention

[0010] To address the shortcomings of the existing technologies, the purpose of this invention is to develop a standardized, low-contamination, high-cell-activity, and stable method for isolating and culturing chicken gonad-derived PGCs, in order to meet the practical application needs of chicken gene editing and germplasm resource conservation.

[0011] To achieve the above objectives, the present invention provides, in one aspect, a method for establishing a lineage of primordial germ cells derived from chicken gonads, wherein, The method for establishing the system includes the following steps: Step 1): Obtain fertilized eggs that have been incubated to stage HH32-35, and aseptically collect chicken embryo gonadal tissue; Step 2): Separation of PGCs; The chicken embryo gonadal tissue is placed in a separation device for collecting primordial germ cells; The separation device includes a container and a sieve with a pore size of 50-100 μm disposed inside the container; The sieve divides the container into two interconnected chambers, upper and lower. The chicken embryo gonadal tissue is placed on the sieve and located in the upper chamber; the container contains a separation culture medium, and the separation culture medium submerges the chicken embryo gonadal tissue; the chicken embryo gonadal tissue is immersed in the separation culture medium for at least 36 hours; then, the culture medium at the bottom of the lower chamber is collected, centrifuged, the supernatant is discarded, and the bottom cells are collected; The isolation culture medium is based on the complete culture medium of poultry PGCs, with calcium chloride added to a final concentration of 3-7 mM; Step 3): In vitro culture of PGCs; The cells collected in Step 2) above are resuspended in avian PGC complete culture medium to prepare a single-cell suspension, and then seeded into culture plates for routine culture, with the medium changed every 2 days; During the culture process, contaminated cell clusters are removed, and cells or cell clusters that meet the PGC morphology are retained until the number of PGC cells accounts for more than 90%, and then cells that meet the PGC morphology are collected for identification; After identification as PGCs, uncontaminated culture wells are selected for continued culture until stable PGC clones appear; Step 4): Establishment of the cell line; use poultry PGCs complete culture medium for subculturing until the PGCs monoclonal cell line is successfully established.

[0012] In some embodiments of the present invention, the aperture of the sieve is 70 μm ± 3 μm.

[0013] In some embodiments of the present invention, in step 2), the separation culture medium is supplemented with calcium chloride to a final concentration of 5 mM, based on the complete culture medium of poultry PGCs.

[0014] In some embodiments of the present invention, the identification is performed by immunofluorescence staining and / or RT-PCR to identify PGCs-specific markers.

[0015] In some embodiments of the present invention, the container is a 50ml conical centrifuge tube.

[0016] In some embodiments of the present invention, the specific operation of the passage culture in step 4) is as follows: when the PGCs density in the culture plate reaches 80%-90%, passage is performed; the culture medium is gently aspirated by pipetting, centrifuged at 2300 rpm for 5 minutes, the supernatant is discarded, the cells are resuspended in fresh culture medium, and seeded into a new cell culture plate at a ratio of 1:2 or 1:3, and culture is continued.

[0017] In another aspect, the present invention provides primordial germ cells derived from chicken gonads obtained by the above-described method of establishing a lineage, wherein the primordial germ cells derived from chicken gonads are male or female primordial germ cells.

[0018] The present invention also provides the application of the above-mentioned chicken gonadal-derived primordial germ cells in gene editing, biodesign breeding, model animal production, or germplasm resource preservation.

[0019] Traditional methods for isolating PGCs from chicken embryo gonads rely on proteolytic enzyme digestion and complex purification procedures (such as multiple centrifugation washes, complex gradient centrifugation, or expensive flow cytometry cell sorting).

[0020] However, the present invention achieves the separation of PGCs from gonadal tissue blocks through a sieve separation device with a specific aperture and a specific separation culture medium, and achieves in-situ purification while collecting PGCs; then, stable PGC clones are obtained through an in vitro culture process.

[0021] The present invention eliminates the digestion step that cannot be bypassed in existing technologies, thus avoiding the destruction of key functional proteins on the cell membrane surface (such as homing receptors and adhesion molecules). PGCs obtained through this method retain their intact cell membrane structure and signal receptors, significantly enhancing their ability to home to and colonize receptor gonads in vivo and ultimately develop into functional gametes. This solves the problem of incomplete functionality in existing PGCs, providing a high-quality cell source for critical applications requiring cell transplantation, such as chicken gene editing and chimera preparation. Furthermore, it significantly reduces subsequent centrifugation and complex sorting procedures, minimizing secondary damage to cells caused by physical stress and improving cell survival rate and initial activity.

[0022] Using the method of this invention, the establishment time of chicken PGCs can be effectively shortened, especially the establishment time of female chicken PGCs; and the establishment success rate is significantly improved (for example, from less than 20% to more than 75%), effectively breaking through the bottleneck restricting the research and application of avian reproductive cells.

[0023] The method of this invention only requires conventional cell culture equipment, is simple to operate, has good reproducibility, and significantly reduces costs, making it easier to promote and apply in ordinary laboratories and production environments.

[0024] The method of establishing a lineage in this invention provides a more stable and suitable method for establishing chicken primordial germ cells, especially female PGCs, which are more difficult to proliferate in vitro. Attached Figure Description

[0025] Figure 1 A schematic diagram of the gonads of a chicken embryo; Figure 2 This is a diagram of the male and female chicken embryo gonads; Figure 3 This is a schematic diagram of the appearance of a separation device used for separating gonadal tissue; Figure 4These are images of PGCs (derived from male chicken embryos) at different stages of their growth process. Figure 5 These are images of PGCs (derived from female chicken embryos) at different stages of their growth process, showing cell morphology at various points in time. Figure 6 The expression of PGC-specific genes was detected by RT-PCR. Figure 7 This involves indirect immunofluorescence staining to detect the expression of PGC-specific proteins. Detailed Implementation

[0026] The technical solution of the present invention will be described in detail below with reference to embodiments, but this does not limit the present invention to the scope of the embodiments described. Process parameters not specified in the embodiments of this application can be performed according to conventional methods, and all raw materials used can be obtained through commercial channels.

[0027] Experimental materials 1. Instruments and equipment: Yongxing 440-cell dual-temperature and dual-triple-control fully automatic incubator, Jiahe 36-cell large-screen desktop constant temperature and humidity incubator, SOPTOP DMSZ8 stereo microscope, Yanan XD-301-150W medical cold light source, Thermo Scientific Multiskan GO microplate reader, Eppendorf 5810R desktop centrifuge, 37℃ / 5%CO2 constant temperature incubator.

[0028] 2. Consumables and reagents: 10cm hookless medical ophthalmic curved forceps, 10cm pointed straight-tipped medical scissors, KDL 1mL sterile syringe with needle, 10cm diameter bacterial culture dish, Sangon Biotech F503316-0001 7cm diameter filter paper, Thermo Fisher Scientific A1110501 StemPro. Accutase digestion solution, KO-DMEM medium (Gibco, catalog number: 10829018), fetal bovine serum (HyClone, catalog number: SH30070.03), chicken serum (HyClone, catalog number: SH30088.03), GlutaMAX-I (Gibco, catalog number: 35050061), non-essential amino acids (Gibco, catalog number: 11140050), nucleoside mixture (Sigma, catalog number: N3667), β-mercaptoethanol (Sigma, catalog number: M6250), LIF (PeproTech, catalog number: 250-02), SCF (PeproTech, catalog number: 450-33), bFGF (PeproTech, catalog number: 100-18B), penicillin-streptomycin (Gibco, catalog number: 15140122), mouse anti-chicken SSEA-1 monoclonal antibody (SANTA). CRUZ (Catalog No.: sc-21702), Rabbit anti-chicken DAZL polyclonal antibody (ProteinTech, Catalog No.: 10946-1-AP), FITC-labeled goat anti-mouse IgG (Beyotime, Catalog No.: A0568), FITC-labeled goat anti-rabbit IgG (Beyotime, Catalog No.: A0562), DAPI staining solution (Sigma, Catalog No.: D9542), TaKaRa RNA extraction kit (Catalog No.: 9767), TaKaRa AMV reverse transcription kit (Catalog No.: D2639), TaKaRa Premix Ex Taq (Catalog No.: RR003A).

[0029] 3. Experimental sample: Fresh fertilized eggs from Sea Blue White chickens.

[0030] Example 1

[0031] Step 1): Obtain fertilized eggs that have been incubated to stage HH32-35, and aseptically collect chicken embryo gonadal tissue; 1a) Hatching and pretreatment of fertilized eggs (I) Take at least 80 fresh Hy-Blue chicken fertilized eggs and wipe them three times along the length of the eggshell with a 75% alcohol swab to remove surface dirt (feces, feed residue, etc.); since they need to be incubated immediately, there is no need for low-temperature storage; (II) Place the fertilized eggs stably on the tray of the Yongxing 440 fully automatic incubator, set the incubation parameters: temperature 37.8℃, humidity 60%, egg turning frequency once every 2 hours, and start the incubation program; (III) When the eggs have been incubated for 8.5 days (corresponding to the chicken embryo development stage of about HH32-35), transfer the fertilized eggs to a Jiahe tabletop constant temperature incubator that has been preheated for 30 minutes (37.8℃, 60% humidity) and let them stand for 10 minutes before use.

[0032] 1b) Aseptic collection procedure of chicken embryo gonadal tissue (1) In the clean bench, wipe the shell of each fertilized egg again with 75% alcohol cotton, gently tap the middle of the eggshell with the end of sterile tweezers (the crack length is about 2cm), cut the eggshell along the crack with sterile scissors, and transfer the contents of the egg (including the chicken embryo) to the sterile waste liquid tank. (2) Carefully tear open the yolk membrane on the surface of the chicken embryo with sterile curved tweezers, pick up the chicken embryo and transfer it to a 10cm bacterial culture dish lined with sterile filter paper. The filter paper absorbs the excess yolk on the surface of the chicken embryo. (3) Place the culture dish on the stage of the stereomicroscope, turn on the cold light source, adjust the chicken embryo so that its abdomen is facing upwards, and use sterile scissors to make an incision of about 1 cm along the midline of the abdomen. Use curved forceps to separate the intestines and liver to expose the kidneys and gonads (see Figure 1 ); (4) Observe the morphology of the gonads: If the gonads of the chicken embryo are symmetrical on both sides, it is determined to be male, and both gonads are selected; if the gonads on the left side of the chicken embryo are larger than those on the right side, it is determined to be female, and both gonads are selected; Figure 2 As shown; (5) Place the gonads of each chicken embryo into a 1.5 mL centrifuge tube containing 150 μL of complete PGC culture medium. Before collecting the next gonad, rinse the syringe needle with DPBS 3 times.

[0033] Step 2): Separation of PGCs; In some embodiments of the present invention, PGCs are separated using a special separation device; the separation device includes a container and a sieve with a pore size of 50-100 μm disposed inside the container; the sieve divides the container into two interconnected chambers, upper and lower.

[0034] Specifically in this embodiment, such as Figure 3 As shown, the container uses 50ml pointed-bottom centrifuge tubes commonly used in laboratories; the flange of the sieve component is mounted on the inlet of the pointed-bottom centrifuge tube, and the sieve part of the sieve component (not specifically shown in the figure) is located inside the container with an aperture of about 70μm; the sieve divides the internal chamber of the container into two connected chambers (connected by the sieve); the container contains separation culture medium, and the liquid level of the separation culture medium submerges the sieve and the chicken embryo gonadal tissue described below; (2a) The chicken embryo gonadal tissue obtained by the above steps (without digestion) is placed on a sieve in the upper chamber and immersed in the separation culture medium for at least 36 hours.

[0035] In some embodiments of the present invention, the isolation culture medium is prepared by adding calcium chloride to a complete culture medium for poultry PGCs, with a final concentration of 3-7 mM. Specifically, in this embodiment, the calcium chloride concentration of the isolation culture medium is 5 mM calcium chloride, and the liquid level is higher than the sieve to achieve immersion of chicken embryo gonadal tissue; then the entire device is placed in a 37°C, 5% CO2 incubator and allowed to stand for induction for at least 36 hours.

[0036] In some embodiments of the present invention, the complete culture medium for PGCs can be a conventional poultry PGCs complete culture medium formulation in the art.

[0037] Specifically, in this embodiment, the formulation of the complete culture medium for PGCs is shown in Table 1 below: Table 1 During the resting period, primordial germ cells (PGCs) in the gonadal tissue of chicken embryos are immersed in a high Ca2+ environment. 2+ In a high concentration of culture medium, calcium ion-mediated cell adhesion regulation causes PGCs to spontaneously migrate out of the gonadal tissue and fall through the sieve pores (70 μm in diameter) into the lower chamber of the centrifuge tube, where they collect at the bottom of the centrifuge tube.

[0038] (2b) After induction, carefully remove the sieve parts and residual tissue, and collect the cell suspension at the bottom of the centrifuge tube; the recommended centrifugation speed for PGCs is 2000-2800 rpm for 5 min; specifically in this embodiment, centrifuge at 2500 rpm for 5 minutes; discard the supernatant and collect the bottom cells.

[0039] Step 3) In vitro culture and establishment of PGCs (3a) For the bottom cells collected in step 2) above, add 200 μL of complete PGC culture medium (without calcium chloride) to each tube, pipette 10 times to make a single cell suspension, and seed it into one well of a 24-well culture plate (supplement with complete PGC culture medium, total volume of 600 μL per well). (3b) After inoculation, the culture plates were placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. The medium was changed every 2 days. 300 μL of the upper culture medium was aspirated and discarded. 300 μL of fresh culture medium (same as above, complete culture medium for PGCs) was added to resuspend the cells and then added back to the original wells. During the culture process, the cell morphology was observed daily under an inverted microscope. Contaminated cell clusters were removed, and PGCs with regular morphology, strong refractive index, and round or oval shape (cells that conform to the PGC cell morphology) were retained. After changing the medium at least 3 times, it was observed whether the number of cells conforming to the PGC cell morphology was the majority. If the number of PGC cells in the field of view was more than 90%, the cells were collected for identification of PGC-specific markers using the following immunofluorescence staining and RT-PCR methods. For those identified as PGCs, select uncontaminated culture wells (clear culture medium, no turbidity) for continued culture until stable PGC clones appear.

[0040] (3c) Identification of the purity and characteristics of PGCs 3c-1, Indirect Immunofluorescence Detection Immunofluorescence staining was performed using PGC-specific markers (such as SSEA-1 and DAZL).

[0041] Take the male and female PGCs obtained above and wash them gently twice with sterile DPBS for 5 minutes each time; Add 4% paraformaldehyde solution and fix at room temperature for 30 minutes; discard the fixative and wash three times with DPBS containing 5% fetal bovine serum (FBS), 5 minutes each time; Add 0.2% Triton X-100 solution and permeabilize on ice for 20 minutes; discard the permeabilization solution and wash three times with DPBS containing 5% FBS for 5 minutes each time; Add 10% FBS to DPBS blocking buffer and incubate at room temperature for 30 minutes. Discard the blocking solution and add the following primary antibodies: mouse anti-chicken SSEA-1 monoclonal antibody (dilution ratio 1:200) and rabbit anti-chicken DAZL polyclonal antibody (dilution ratio 1:150). Incubate overnight at 4°C. Discard the primary antibodies and wash three times with DPBS containing 5% FBS for 5 minutes each time. Secondary antibodies were added separately: CY5-labeled goat anti-mouse IgG (dilution ratio 1:500) and CY3-labeled goat anti-rabbit IgG (dilution ratio 1:500), and incubated at room temperature in the dark for 1 hour; the secondary antibodies were discarded, and the cells were washed 3 times with DPBS containing 5% FBS for 5 minutes each time; Add DAPI staining solution (concentration 1 μg / mL) and stain nuclei at room temperature in the dark for 5 minutes. The slide was mounted with an anti-fluorescence quencher and observed under a fluorescence microscope.

[0042] If cells exhibit specific fluorescence (SSEA-1, appearing as red fluorescence) and the cell nucleus appears blue (DAPI staining), they are identified as target PGCs. Results are as follows... Figure 7 As shown.

[0043] from Figure 7 As can be seen, under a fluorescence microscope, more than 95% of the cells in both female and male samples were positive for SSEA-1, proving that the obtained female and male PGCs had high purity.

[0044] 3c-2, RT-PCR identification (detection of specific genes in PGCs) mRNA was extracted from each sample and reverse transcribed into cDNA. Using cDNA as a template, PGC-specific expression genes (Nanog, SOX2, DDX4, DAZL, TERT) were amplified by PCR, with GAPDH as an internal control. The PCR products were analyzed by agarose gel electrophoresis to detect the expression of each gene. Results are shown below. Figure 6 As shown.

[0045] (3d) Subculture When the PGCs density in the culture plate reaches 90%, passage is performed; the culture medium is gently aspirated by pipetting, centrifuged at 2300 rpm for 5 minutes, the supernatant is discarded, the cells are resuspended in fresh culture medium, and seeded into a new cell culture plate at a ratio of 1:2 or 1:3, and culture is continued; in the embodiments of this application, after 5-8 consecutive passages, a stable PGCs cell line is obtained.

[0046] Experimental results In the inventors' experiments, small PGC clones (approximately 5-10 cells / clone) appeared in the wells derived from male chicken embryos starting around day 7. By day 15, all 20 PGCs derived from male chicken embryos had formed stable clones, were passaged, and the line was successfully established; see [link to relevant documentation]. Figure 4 ; Clones appeared later in the female wells, with small PGCs clones appearing around day 16; by day 25, 15 out of 20 PGCs derived from female chicken embryos had formed stable clones, ready for passage, and the line was successfully established; see [link to relevant documentation]. Figure 5 In the prior art, it is difficult to proliferate female PGCs in vitro, and the establishment time for female chicken PGCs usually takes 35-50 days; however, using the method of the present invention, it only takes about 22-25 days, which can significantly shorten the establishment time.

[0047] This experiment included a control group, with the experimental group and control group randomly selected from the same batch of fresh Sea Blue chicken fertilized eggs; The experimental group (male / female) underwent PGC cell isolation and line establishment according to the method described in the above embodiments; the control group (male / female) used the first method in the background art (mechanically dissecting and digesting the gonads) for PGC cell isolation and line establishment (400 μL of BSA-PBS buffer was added to the isolated gonadal tissue, followed by 100 μL of 0.25% trypsin-EDTA digestion solution, adjusting the final concentration of trypsin-EDTA in the system to 0.05%; the treated gonadal samples were placed at 37.5℃ with a volume fraction of 5%). Incubate in a CO2 constant temperature environment for 8 min; gently pipette the gonadal tissue 30-40 times to mechanically dissociate and disperse the gonadal tissue clumps; immediately add an equal volume of cell culture medium containing fetal bovine serum (FBS) to terminate the trypsin digestion reaction; add PBS buffer to the reaction system to a final volume of 4.5 mL, and centrifuge at 2500 rpm for 5 min at room temperature; discard the supernatant after centrifugation, resuspend the cell pellet in PGC complete culture medium, and transfer the resuspended cells to 24-well culture plates for further culture. The experimental results are shown in Table 2 below.

[0048] Table 2 As can be seen from Table 2, using the method of this embodiment, the success rate of establishing male lines reached 100%, and the success rate of establishing female lines reached 75%, both of which were significantly higher than their respective control groups under the same conditions.

[0049] This invention is not limited to the above-described embodiments. Any changes in shape or structure are within the scope of protection of this invention. The scope of protection of this invention is defined by the appended claims. Those skilled in the art can make various changes, modifications, substitutions, combinations, and simplifications to these embodiments without departing from the principles and essence of this invention. All such changes and simplifications should be considered equivalent substitutions and fall within the scope of protection of this invention.

Claims

1. A method for establishing a lineage of primordial germ cells derived from chicken gonads, characterized in that, The method for establishing the system includes the following steps: Step 1): Obtain fertilized eggs that have been incubated to stage HH32-35, and aseptically collect chicken embryo gonadal tissue; Step 2): Separation of PGCs; The chicken embryo gonadal tissue is placed in a separation device for collecting primordial germ cells; The separation device includes a container and a sieve with a pore size of 50-100 μm disposed inside the container; The sieve divides the container into two interconnected chambers, upper and lower. The chicken embryo gonadal tissue is placed on the sieve and located in the upper chamber; the container contains a separation culture medium, and the separation culture medium submerges the chicken embryo gonadal tissue; the chicken embryo gonadal tissue is immersed in the separation culture medium for at least 36 hours; then, the culture medium at the bottom of the lower chamber is collected, centrifuged, the supernatant is discarded, and the bottom cells are collected; The isolation culture medium is based on the complete culture medium of poultry PGCs, with calcium chloride added to a final concentration of 3-7 mM; Step 3): In vitro culture of PGCs; The cells collected in Step 2) above are resuspended in avian PGC complete culture medium to prepare a single-cell suspension, and then seeded into culture plates for routine culture, with the medium changed every 2 days; During the culture process, contaminated cell clusters are removed, and cells or cell clusters that meet the PGC morphology are retained until the number of PGC cells accounts for more than 90%, and then cells that meet the PGC morphology are collected for identification; After identification as PGCs, uncontaminated culture wells are selected for continued culture until stable PGC clones appear; Step 4): Establishment of the cell line; use poultry PGCs complete culture medium for subculturing until the PGCs monoclonal cell line is successfully established.

2. The method for establishing a system as described in claim 1, characterized in that: The sieve has an aperture of 70μm±3μm.

3. The method for establishing a system as described in claim 1, characterized in that: In step 2), the separation culture medium is prepared by adding calcium chloride to a final concentration of 5 mM, based on the complete culture medium of poultry PGCs.

4. The method for establishing a system as described in claim 1, characterized in that: The identification was performed using immunofluorescence staining and / or RT-PCR to identify PGCs-specific markers.

5. The method for establishing a system as described in any one of claims 1 to 4, characterized in that: The container is a 50ml pointed-bottom centrifuge tube.

6. The method for establishing a system as described in any one of claims 1 to 4, characterized in that: The specific operation of the passage culture in step 4) is as follows: when the PGCs density in the culture plate reaches 80%-90%, passage is performed; the culture medium is gently aspirated by pipetting, centrifuged at 2300 rpm for 5 minutes, the supernatant is discarded, the cells are resuspended in fresh culture medium, and seeded into a new cell culture plate at a ratio of 1:2 or 1:3, and culture is continued.

7. Primitive germ cells derived from chicken gonads obtained by the establishment method according to any one of claims 1 to 6, characterized in that: The primordial germ cells derived from chicken gonads are either male or female primordial germ cells.

8. The application of the primordial germ cells derived from chicken gonads as described in claim 7 in gene editing, biodesign breeding, model animal production, or germplasm resource preservation.

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