Method for building cell line through serum-free culture of poultry primordial germ cells

Abstract

The invention discloses a method for establishing a cell line by serum-free culture of PGCs (Primordial Germ Cells) of breeding poultry, the cell line is prepared by blending calcium-free DMEM (Dulbecco Modified Eagle Medium), water and Knockout-DMEM, the cell line is more suitable for osmotic pressure and calcium ion concentration required by proliferation of the PGCs of the breeding poultry, various components of serum-free culture are perfected by using fresh egg white, and the cell line is more suitable for proliferation of the PGCs of the breeding poultry. The combination of growth factors required by primordial germ cells is optimized by adding the FGF-1, so that 100% of cell line establishment efficiency and rapid growth of cells from reproduction crescent, blood and gonad tissues of chicken embryos are realized, and a technical basis is provided for poultry frozen breed conservation, stem cell propagation and gene editing operation.

Description

Technical Field

[0001] The present invention relates to a method for establishing a cell line by serum-free culture of avian primordial germ cells, and belongs to the field of agricultural biotechnology. Background Art

[0002] As precursors of germ cells, primordial germ cells (PGCs) have the potential to differentiate into sperm and eggs, and are key tools for poultry genetic improvement and transgenic technology research. Through in vitro culture amplification and cryopreservation techniques, PGCs can be used to cultivate and expand a large number of avian populations with excellent traits, thereby improving the production efficiency and economic benefits of the poultry industry.

[0003] Studies have shown that serum-free culture of chicken PGCs can be achieved by adjusting the composition of the culture medium (such as adding specific growth factors, cytokines, nutrients, specific osmotic pressure conditions, etc.). In 2015, researchers such as Whyte referred to the serum-free culture study of mammalian embryonic primordial germ cells. In the in vitro culture system of chicken PGCs, by customizing a culture medium suitable for the osmotic pressure of chicken PGC cells and adding various nutritional components and cytokines such as B27 additive, nucleosides, fresh egg white, sodium heparin, and recombinant human activin A, a PGC cell line without serum and feeder cells was obtained (Michael J. McGrew, etc. FGF, Insulin, and SMAD Signaling Cooperate for Avian Primordial Germ Cell Self-Renewal. Stem Cell Reports. Vol. 5, 1171–1182). Compared with the method containing serum and feeder cells, this method has the advantages of relatively simple operation, reduced probability of exogenous contamination, and improved gene editing efficiency. However, the culture medium of the above method needs to be customized and is expensive and difficult to apply. In 2023, researchers such as Michael J. McGrew disclosed an alternative formula for customizing the culture medium in their work. By adding a certain proportion of cell culture water to DMEM without calcium ions and adding a 0.15 mM calcium chloride solution, the effect of reducing the osmotic pressure of the culture medium was achieved. (Idoko-Akoh, A., & McGrew, M. J. (2023). Generation of Genome-Edited Chicken Through Targeting of Primordial Germ Cells. In Methods in Molecular Biology (Vol. 2631, pp. 419-441). Article 2 Methods in molecular biology) Subsequently, some researchers have adopted this method by adding water (volume ratio of DMEM without calcium ions to water is 3:1) and successfully obtained the PGC cell line (Ibrahim, M.; Grochowska, E.; Lázár, B.; Várkonyi, E.; Bednarczyk, M.; Stadnicka, K. The Effect of Short-and Long-Term Cryopreservation on Chicken Primordial Germ Cells. Genes 2024, 15, 624.).

[0004] Although certain progress has been made in the serum-free culture of avian PGCs, the following technical problems still remain:

[0005] 1) It is difficult to achieve 100% success in establishing cell lines with existing avian PGC culture methods, which makes it difficult to successfully establish and preserve cell lines of some precious avian breeds.

[0006] 2) Existing avian PGC culture methods are more suitable for the amplification of male avian cell lines, while the amplification of female avian cell lines still has certain difficulties and the success rate of establishing cell lines is low.

[0007] 3) The cells in the existing culture methods grow slowly and it takes a long time to establish cell lines, which is difficult to meet the urgent needs of the current poultry breeding industry for the preservation and propagation of germplasm resources.

[0008] 4) The existing culture system has a large number of components, there are some non-essential components, and it has not been optimized to the best conditions. Summary of the Invention

[0009] In view of the above deficiencies of the prior art, the present invention provides a method for establishing a cell line by serum-free culture of avian primordial germ cells, aiming to solve the technical problems in the prior art of lacking a cell line establishment method with a high success rate of cell line establishment, rapid cell growth, and being applicable to the amplification of both male and female avian cell lines at the same time.

[0010] The first technical solution provided by the present invention is a culture system for avian primordial germ cells. The culture system includes a basal medium composed of calcium-free DMEM and Knockout-DMEM, and a supplementary component and a cytokine are added to the basal medium. The supplementary component includes B27, glutamine, non-essential amino acids, β-mercaptoethanol, nucleosides, sodium pyruvate, 1% fresh egg white, and sodium heparin. The cytokine includes chicken serum, activin A, acidic fibroblast growth factor (FGF-1), and basic fibroblast growth factor (FGF-2).

[0011] Based on the above literature research, the present invention uses a certain proportion of KO-DMEM medium as a calcium source and a supplement of nutritional components, improves the formula and optimizes the components, and solves the problem that adding water will cause the dilution of nutritional components. The new formula adopted by the present invention can achieve a 100% cell line establishment efficiency, and the use of fresh egg white is also not seen in other prior responses.

[0012] In some embodiments, the basal medium is composed of 69% DMEM, 8% Knockout-DMEM, and 23% cell culture water.

[0013] In some embodiments, the supplementary components include 1×B27 supplement, 2 mM glutamine, 1×non-essential amino acids, 0.1 mM β-mercaptoethanol, 1×nucleosides, 1 mM sodium pyruvate, 1% fresh egg white, and 0.1 mg / mL sodium heparin.

[0014] In some embodiments, the cytokines include 0.2% chicken serum, 50 ng / mL activin A, 50 ng / mL FGF-1, and 10 ng / mL FGF-2.

[0015] In some embodiments, the culture system further includes antibiotics, and the antibiotics include penicillin-streptomycin and streptomycin.

[0016] Further, the antibiotic is 0.2×penicillin-streptomycin.

[0017] The second technical solution provided by the present invention is a method for establishing a cell line of avian primordial germ cells. The method is to culture avian primordial germ cells using the culture system described in the first technical solution. The cell line establishment criteria are as follows: the cell number reaches 2×10 6 cells, and they are derived from a single embryo.

[0018] In some embodiments, the avians are chickens, ducks, geese, quails, pigeons, turkeys, or wild birds.

[0019] Further, the avian is a chicken. Optionally, the chicken is a breeding chicken. Optionally, the breeding chicken is a broiler breeding chicken, a laying hen breeding chicken, or a local breed chicken.

[0020] In some embodiments, the avian primordial germ cells are derived from the germinal crescent tissue, blood, or gonadal tissue.

[0021] In some embodiments, the method for collecting the germinal crescent tissue is as follows: at the 5th to 10th stage of avian embryo development, the embryo is taken out, the germinal crescent area is found under a microscope, and the germinal crescent tissue is separated with ophthalmic scissors and forceps.

[0022] In some embodiments, the method for collecting the blood is as follows: 1-2 μL of blood is collected from the heart or peripheral blood vessels of avian embryos hatched to the 15th to 16th stage.

[0023] In some embodiments, the method for collecting the gonadal tissue is as follows: the avian embryo is hatched to 7 days, the embryo is taken out, the gonad of the embryo is found under a microscope, and the gonad is separated with ophthalmic scissors and forceps.

[0024] In some embodiments, the culture conditions are: culturing under the conditions of 37°C and 5% CO2.

[0025] The third technical solution provided by the present invention is the application of the culture system described in the first technical solution or the method described in the second technical solution in the expansion and propagation of poultry.

[0026] The fourth technical solution provided by the present invention is the application of the culture system described in the first technical solution or the method described in the second technical solution in the field of poultry germplasm conservation or gene editing.

[0027] Beneficial effects

[0028] 1) Improve the culture efficiency and achieve a 100% success rate in establishing cell lines. It provides high-efficiency technical support for the preservation and expansion of endangered and precious poultry germplasm resources.

[0029] 2) This method solves the problem that the amplification of female poultry cell lines is prone to failure, thus realizing the stable and efficient establishment of poultry PGC cell lines without being restricted by the gender of poultry individuals.

[0030] 3) This method has a fast cell amplification speed, greatly shortening the time required for establishing cell lines. It is beneficial for gene editing operations and provides a high-quality and efficient cell line source and a reliable technical platform for the subsequent preparation of transgenic chickens. Description of the drawings

[0031] Figure 1 Morphology of cells cultured from the genital crescent tissue; A shows cells cultured from the genital crescent in the experimental group for 2 days; B shows cells cultured from the genital crescent in the experimental group for 9 days; C shows cells cultured from the genital crescent in the experimental group for 20 days.

[0032] Figure 2 Morphology of cells cultured from blood; A shows cells cultured from blood in the experimental group for 2 days; B shows cells cultured from blood in the experimental group for 9 days; C shows cells cultured from blood in the experimental group for 20 days.

[0033] Figure 3 Morphology of cells cultured from gonads; A shows cells cultured from gonads in the experimental group for 2 days; B shows cells cultured from gonads in the experimental group for 9 days; C shows cells cultured from gonads in the experimental group for 20 days.

[0034] Figure 4 It is a comparison chart of cell growth curves. Detailed implementation manners

[0035] The following are descriptions of the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0036] Raw materials used in the embodiments:

[0037] Basal medium composition: 69% DMEM (Gibco, calcium-free), 8% Knockout-DMEM (Gibco), 23% cell culture water (Beyotime).

[0038] Added components: 1×B27 supplement (Beyotime), 2 mM glutamine, 1×non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1×nucleosides (Merck), 1 mM sodium pyruvate (Gibco), 1% fresh egg white (extracted from SPF eggs), 0.1 mg / mL heparin sodium (Sigma), 0.2×penicillin-streptomycin.

[0039] Cytokines: 0.2% chicken serum (Sigma), activin A 50 ng / mL, FGF-1 50 ng / mL, recombinant human FGF-2 10 ng / mL.

[0040] Among them, activin A is recombinant human activin A (Novoprotein), FGF-1 is recombinant human FGF-1 (Novoprotein), and FGF-2 is recombinant human FGF-2 (Novoprotein).

[0041] Fresh egg white is extracted from SPF eggs, that is, under sterile conditions, the eggshell is opened, and the thin egg white is drawn with a syringe, stored at -20°C, and thawed for use.

[0042] Example 1 Construction of the cell line of PGCs obtained from the genital crescent

[0043] 1) Experimental group:

[0044] Basal medium composition: 69% DMEM (Gibco, calcium-free), 8% Knockout-DMEM (Gibco), 23% cell culture water (Beyotime)

[0045] Added components: 1×B27 supplement (Beyotime), 2 mM glutamine, 1×non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1×nucleosides (Merck), 1 mM sodium pyruvate (Gibco), 1% fresh egg white (extracted from SPF eggs), 0.1 mg / mL heparin sodium (Sigma), 0.2% penicillin / streptomycin.

[0046] Cytokines: 0.2% chicken serum (Sigma), recombinant human activin A 50 ng / mL, recombinant human FGF-1 50 ng / mL, recombinant human FGF-2 10 ng / mL.

[0047] 2) Control group 1 (without fresh egg white, without Knockout-DMEM, with CaCl2)

[0048] Composition of the basal medium: 75% DMEM (Gibco, without calcium ions), 25% cell culture water (Beyotime).

[0049] Added components: 1×B27 supplement (Beyotime), 2 mM glutamine, 1×non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1×nucleosides (Merck), 1 mM sodium pyruvate (Gibco), 1% ovalbumin (powder, Sigma), 0.1 mg / mL heparin sodium (Sigma), 0.2% penicillin / streptomycin, and 0.15 mM CaCl2 was added.

[0050] Cytokines: 0.2% chicken serum (Sigma), recombinant human activin A 50 ng / mL, recombinant human FGF-2 10 ng / mL.

[0051] 3) Control group 2 (with serum and feeder cells)

[0052] 46% KO-DMEM, 40% conditioned medium of rat hepatocytes (Buffalo Rat Liver BRL) in KO-DMEM, 7.5% fetal bovine serum, 1% glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 0.1% β-mercaptoethanol, 1% penicillin / streptomycin.

[0053] Cytokines: 2.5% chicken serum, recombinant human FGF-1 50 ng / mL, recombinant human FGF-2 10 ng / mL.

[0054] 2. Establishment of cell line

[0055] Using fresh white - feather broiler hatching eggs as experimental materials

[0056] 1) At the 5th - 10th day of chicken embryo development, the embryo was taken out, the germinal crescent region was found under the microscope, and the germinal crescent tissue was separated with ophthalmic scissors and forceps and placed into a centrifuge tube containing 300 μL of PGC culture medium.

[0057] 2) The separated germinal crescent tissue was transferred to a 48 - well cell culture plate, complete medium was added, and it was pipetted several times with a pipette gun to disperse the tissue into a single - cell suspension.

[0058] 3) Cultured under the conditions of 37°C and 5% CO2, and the culture medium was replaced with fresh medium every 48 hours. When the cells grew to confluence, they were passaged at a density of 1×10 5 cells / mL (for the control group of serum - containing cell culture, irradiated rat hepatocytes were added as the feeder layer).

[0059] 4) Regularly collect cell counts and record the number of days required for the cell growth quantity to reach 2×10 6 cells, which is the number of days for establishing the cell line.

[0060] The results are shown in Table 1 and Figure 1 as follows.

[0061] Table 1 Establishment efficiency of PGC cell lines derived from the germinal crescent of breeder chickens

[0062] Number of embryos Cell coefficient Number of days for establishing cell line (average) Establishment efficiency of cell line Experimental group 14 14 21～25(22) 100％ Control group 1 10 8 22～26(23) 80％ Control group 2 10 4 45～60(50) 40％

[0063] The result data shows that the establishment efficiency of the experimental group is significantly higher than that of Control Groups 1 and 2. The number of days required for establishing the cell line is significantly shorter than that of Control Group 2 (with serum and feeder cells). Moreover, from the perspective of cell morphology, the cells do not form clusters and are single suspended cells.

[0064] Example 2 Construction of PGC cell lines obtained from blood

[0065] 1. Experimental grouping

[0066] 1) Experimental group:

[0067] Composition of the basal medium: 69% DMEM (Gibco, calcium-free), 8% Knockout-DMEM (Gibco), 23% cell culture water (Beyotime)

[0068] Added components: 1×B27 supplement (Beyotime), 2 mM glutamine, 1×non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1×nucleosides (Merck), 1 mM sodium pyruvate (Gibco), 1% fresh egg white (extracted from SPF eggs), 0.1 mg / mL heparin sodium (Sigma), 0.2% penicillin / streptomycin.

[0069] Cytokines: 0.2% chicken serum (Sigma), 50 ng / mL recombinant human activin A, 50 ng / mL recombinant human FGF-1, 10 ng / mL recombinant human FGF-2.

[0070] 2) Control group (without fresh egg white, without FGF-1, without Knockout-DMEM, with CaCl2)

[0071] Composition of the basal medium: 75% DMEM (Gibco, calcium-free), 25% cell culture water (Beyotime).

[0072] Added components: 1×B27 supplement (Beyotime), 2 mM glutamine, 1×non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1×nucleosides (Merck), 1 mM sodium pyruvate (Gibco), 1% ovalbumin (powder, Sigma), 0.1 mg / mL heparin sodium (Sigma), 0.2% penicillin / streptomycin, and 0.15 mM CaCl2 was added.

[0073] Cytokines: 0.2% chicken serum (Sigma), recombinant human activin A 50 ng / mL, recombinant human FGF-2 10 ng / mL.

[0074] 2. Establishment of cell line

[0075] Fresh white - feather broiler hatching eggs were used as experimental materials.

[0076] 1) Blood collection: From the heart or peripheral blood vessels at the 15 - 16th stage of incubation to hatching, 1 - 2 μL of blood was collected with a micropipette - controlled micromanipulation needle and transferred to a 48 - well cell culture plate, and complete medium was added.

[0077] 2) Pipette the cells several times to disperse them evenly. Culture under the conditions of 37°C and 5% CO2.

[0078] 3) Replace the culture medium with fresh medium every 48 hours. When the cells grow to confluence, passage them at a density of 100,000 cells / mL.

[0079] Regularly collect cells for counting, and record the number of days required for the cell growth to reach 2×10 6 That is the number of days required for establishing the cell line.

[0080] The results are shown in Table 2 and Figure 2 as follows.

[0081] Table 2 Establishment efficiency of PGC cell line from broiler blood

[0082] Number of embryos Cell coefficient Number of days for establishing cell line (average) Establishment efficiency of cell line Experimental group 8 8 20～25(22) 100％ Control group 10 8 22～30(26) 80％

[0083] The result data shows that the establishment efficiency of the experimental group is significantly higher than that of the control group, the number of days required for establishing the cell line is less than that of the control group, and from the cell morphology, the cells do not form clusters and are single suspended cells.

[0084] Example 3 Construction of PGC cell line from gonadal tissue

[0085] 1. Experimental grouping

[0086] 1) Experimental group:

[0087] Base medium composition: 69% DMEM (Gibco, calcium-free), 8% Knockout-DMEM (Gibco), 23% cell culture water (Beyotime)

[0088] Added components: 1×B27 supplement (Beyotime), 2 mM glutamine, 1×non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1×nucleosides (Merck), 1 mM sodium pyruvate (Gibco), 1% fresh egg white (extracted from SPF eggs), 0.1 mg / mL heparin sodium (Sigma), 0.2% penicillin / streptomycin.

[0089] Cytokines: 0.2% chicken serum (Sigma), recombinant human activin A 50 ng / mL, recombinant human FGF-1 50 ng / mL, recombinant human FGF-2 10 ng / mL

[0090] 2) Control group (without fresh egg white, without FGF-1, without Knockout-DMEM, with CaCl2 added)

[0091] Base medium composition: 75% DMEM (Gibco, calcium-free), 25% cell culture water (Beyotime).

[0092] Added components: 1×B27 supplement (Beyotime), 2 mM glutamine, 1×non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1×nucleosides (Merck), 1 mM sodium pyruvate (Gibco), 1% ovalbumin (powder, Sigma), 0.1 mg / mL heparin sodium (Sigma), 0.2% penicillin / streptomycin, with 0.15 mM CaCl2 added.

[0093] Cytokines: 0.2% chicken serum (Sigma), recombinant human activin A 50 ng / mL, recombinant human FGF-2 10 ng / mL.

[0094] 2. Establishment of cell line

[0095] Using fresh white - feather broiler hatching eggs as experimental materials.

[0096] 1) Incubate chicken embryos until 7 days, then take out the embryos. Locate the gonads of the embryos under a microscope, separate the gonads with ophthalmic scissors and forceps, and collect them into a centrifuge tube.

[0097] 2) Further digest the gonad tissue with trypsin into a single - cell suspension, centrifuge to remove trypsin and add complete medium. Resuspend the cells and transfer them to a 48 - well cell culture plate, and culture them at 37°C and 5% CO2.

[0098] 3) Replace the culture medium every 48 hours. When the cells grow to confluence, passage them at a density of 100,000 cells / mL.

[0099] 4) Regularly collect cells and perform cell counting, and record the number of days required for the cell growth to reach 2×10 6 The number of days required, which is the number of days for establishing the cell line.

[0100] Table 3 Establishment efficiency of PGC from chicken blood

[0101] Number of embryos Cell coefficient Number of days for establishing cell line (average) Establishment efficiency of cell line Experimental group 22 22 14～24(19) 100％ Control group 12 8 18～25(22) 66.7％

[0102] The result data shows that the establishment efficiency of the experimental group is significantly higher than that of the control group, and the number of days required for establishing the cell line is less than that of the control group. From the perspective of cell morphology, the cells do not form clusters and are single suspended cells.

[0103] Example 4 Comparison of growth curves

[0104] 1. Experimental grouping

[0105] 1) Experimental group A (without fresh egg white, with FGF-1):

[0106] Composition of the basal medium: 69% DMEM (Gibco, without calcium ions), 8% Knockout-DMEM (Gibco), 23% cell culture water (Beyotime).

[0107] Added components: 1×B27 supplement (Beyotime), 2 mM glutamine, 1×non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1×nucleosides (Merck), 1 mM sodium pyruvate (Gibco), 0.1 mg / mL heparin sodium (Sigma), 0.2% penicillin / streptomycin.

[0108] Cytokines: 0.2% chicken serum (Sigma), recombinant human activin A 50 ng / mL, recombinant human FGF-1 50 ng / mL, recombinant human FGF-2 10 ng / mL

[0109] 2) Experimental group B (with fresh egg white, without FGF-1, without Knockout-DMEM, with CaCl2).

[0110] Composition of the basal medium: 75% DMEM (Gibco, without calcium ions), 25% cell culture water (Beyotime).

[0111] Additive components: 1×B27 supplement (Beyotime), 2 mM glutamine, 1×non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1×nucleosides (Merck), 1 mM sodium pyruvate (Gibco), 1% fresh egg white (extracted from SPF eggs), 0.1 mg / mL heparin sodium (Sigma), 0.2% penicillin / streptomycin, add 0.15 mM CaCl2.

[0112] Cytokines: 0.2% chicken serum (Sigma), recombinant human activin A 50 ng / mL, recombinant human FGF-2 10 ng / mL.

[0113] 3) Control group C (without fresh egg white, without FGF-1, without Knockout-DMEM, add CaCl2)

[0114] Composition of basal medium: 75% DMEM (Gibco, without calcium ions), 25% cell culture water (Beyotime).

[0115] Additive components: 1×B27 supplement (Beyotime), 2 mM glutamine, 1×non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1×nucleosides (Merck), 1 mM sodium pyruvate (Gibco), 1% ovalbumin (powder, Sigma), 0.1 mg / mL heparin sodium (Sigma), 0.2% penicillin / streptomycin, add 0.15 mM CaCl2.

[0116] Cytokines: 0.2% chicken serum (Sigma), recombinant human activin A 50 ng / mL, recombinant human FGF-2 10 ng / mL.

[0117] 2. Plot the growth curve

[0118] Select the cell line (CC3, cultured in vitro for 22 days) obtained by the culture method of the control group in Example 2. After collecting, counting, centrifuging, and resuspending the cells, they are evenly divided into three groups, respectively labeled as experimental groups A, B, and control group C, with 1.0×10 6 cells in each group.

[0119] Culture under the conditions of 37°C and 5% CO2. Count the cells in each group on the 2nd, 4th, and 5th days after culture, record the cell numbers and plot the growth curve.

[0120] The results are shown in Table 4.

[0121] Table 4 Cell growth counts and growth curve graphs at different culture days

[0122] Day 0 Day 2 Day 4 Day 5 A <![CDATA[1.0×10 6 > <![CDATA[3.4×10 6 > <![CDATA[1.0×10 7 > <![CDATA[1.4×10 7 > B <![CDATA[1.0×10 6 > <![CDATA[2.6×10 6 > <![CDATA[1.0×10 7 > <![CDATA[1.2×10 7 > C <![CDATA[1.0×10 6 > <![CDATA[2.9×10 6 > <![CDATA[6.7×10 6 > <![CDATA[8.6×10 6 >

[0123] The results showed that in experimental group A supplemented with recombinant human FGF1 and experimental group B supplemented with fresh egg white, the cell growth rate was significantly higher than that in control group C without recombinant human FGF1 and fresh egg white. This indicates that supplementing recombinant human FGF1 and fresh egg white can increase the growth rate of PGCs.

[0124] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A poultry primordial germ cell culture system, characterized in that, The culture system includes a basal medium composed of calcium-free DMEM and Knockout-DMEM, and supplementary components and cytokines are added to the basal medium. The supplementary components include B27, glutamine, non-essential amino acids, β-mercaptoethanol, nucleosides, sodium pyruvate, 1% fresh egg white, and sodium heparin. The cytokines include chicken serum, activin A, acidic fibroblast growth factor (FGF-1), and basic fibroblast growth factor (FGF-2).

2. The culture system according to claim 1, wherein The basal medium is composed of 69% DMEM, 8% Knockout-DMEM, and 23% cell culture water.

3. The culture system according to claim 2, characterized in that, The supplementary components include 1×B27 supplement, 2 mM glutamine, 1×non-essential amino acids, 0.1 mM β-mercaptoethanol, 1×nucleosides, 1 mM sodium pyruvate, 1% fresh egg white, and 0.1 mg / mL sodium heparin.

4. The culture system according to claim 1, wherein The cytokines include 0.2% chicken serum, 50 ng / mL activin A, 50 ng / mL FGF-1, and 10 ng / mL FGF-2.

5. A method for establishing a line of avian primordial germ cells, characterized in that, The method is to culture avian primordial germ cells using the culture system according to any one of claims 1 to 5.

6. The method according to claim 5, wherein The avians are chickens, ducks, geese, quails, pigeons, turkeys, wild birds. Optionally, the avian is a chicken.

7. The method according to claim 5, wherein The avian primordial germ cells are derived from the germinal crescent tissue, blood, or gonadal tissue.

8. The method according to claim 5, wherein The culture conditions are: culturing under the conditions of 37°C and 5% CO2.

9. Application of the culture system according to any one of claims 1 to 4 or the method according to any one of claims 5 to 8 in avian propagation.

10. Application of the culture system according to any one of claims 1 to 4 or the method according to any one of claims 5 to 8 in the fields of avian cryopreservation or gene editing.

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