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US-20260125642-A1 - CHICKEN SERUM-FREE MEDIUM FOR PROMOTING PROLIFERATION OF CHICKEN PRIMORDIAL GERM CELLS (PGCs) AND USE OF CHICKEN SERUM-FREE MEDIUM

US20260125642A1US 20260125642 A1US20260125642 A1US 20260125642A1US-20260125642-A1

Abstract

A chicken serum-free medium for promoting proliferation of chicken primordial germ cells (PGCs) and a use of the chicken serum-free medium are provided. In the chicken serum-free medium, ovotransferrin is used instead of chicken serum. The ovotransferrin is used instead of chicken serum in a PGC culture system, and ovotransferrin can keep the self-renewing of PGCs to provide a basis for exploring a stable culture system. The chicken serum-free medium can avoid the adverse effects caused by an unclear composition of chicken serum. In addition, the accurate use of various growth factors can provide excellent culture conditions and reduce the culture cost.

Inventors

  • Qisheng ZUO
  • Xin Liu
  • BiChun Li
  • Wei Gong
  • Xiaoqian Lv
  • Yingjie Niu
  • Kai Jin

Assignees

  • YANGZHOU UNIVERSITY

Dates

Publication Date
20260507
Application Date
20241216

Claims (12)

  1. 1 . A chicken serum-free medium for promoting proliferation of chicken primordial germ cells (PGCs), wherein in the chicken serum-free medium, ovotransferrin is used instead of chicken serum.
  2. 2 . The chicken serum-free medium for promoting the proliferation of the chicken PGCs according to claim 1 , wherein the chicken serum-free medium further comprises a Dulbecco's Modified Eagle Medium (DMEM), ultrafiltration water, and additives; and the additives comprise a concentrated calcium chloride stock solution, a B-27™ additive, a GlutaMax additive, an MEM non-essential amino acids solution, 2-mercaptoethanol, an EmbryoMax® nucleoside, sodium pyruvate, albumin, sodium heparin, basic fibroblast growth factor, activin A, and a penicillin-streptomycin mixed solution.
  3. 3 . The chicken serum-free medium for promoting the proliferation of the chicken PGCs according to claim 1 , wherein a concentration of the ovotransferrin in the chicken serum-free medium is 0.1 mg/mL to 1 mg/mL.
  4. 4 . The chicken serum-free medium for promoting the proliferation of the chicken PGCs according to claim 3 , wherein the concentration of the ovotransferrin in the chicken serum-free medium is 0.5 mg/mL.
  5. 5 . A method for promoting proliferation of chicken PGCs, comprising using the chicken serum-free medium according to claim 1 .
  6. 6 . A method for promoting proliferation of chicken PGCs, comprising the following steps: inoculating the chicken PGCs into the chicken serum-free medium according to claim 1 , and culturing at 37° C. for a predetermined period of time until a required number of the chicken PGCs are produced.
  7. 7 . The method according to claim 5 , wherein the chicken serum-free medium further comprises a Dulbecco's Modified Eagle Medium (DMEM), ultrafiltration water, and additives; and the additives comprise a concentrated calcium chloride stock solution, a B-27™ additive, a GlutaMax additive, an MEM non-essential amino acids solution, 2-mercaptoethanol, an EmbryoMax® nucleoside, sodium pyruvate, albumin, sodium heparin, basic fibroblast growth factor, activin A, and a penicillin-streptomycin mixed solution.
  8. 8 . The method according to claim 5 , wherein a concentration of the ovotransferrin in the chicken serum-free medium is 0.1 mg/mL to 1 mg/mL.
  9. 9 . The method according to claim 8 , wherein the concentration of the ovotransferrin in the chicken serum-free medium is 0.5 mg/mL.
  10. 10 . The method according to claim 6 , wherein the chicken serum-free medium further comprises a Dulbecco's Modified Eagle Medium (DMEM), ultrafiltration water, and additives; and the additives comprise a concentrated calcium chloride stock solution, a B-27™ additive, a GlutaMax additive, an MEM non-essential amino acids solution, 2-mercaptoethanol, an EmbryoMax® nucleoside, sodium pyruvate, albumin, sodium heparin, basic fibroblast growth factor, activin A, and a penicillin-streptomycin mixed solution.
  11. 11 . The method according to claim 6 , wherein a concentration of the ovotransferrin in the chicken serum-free medium is 0.1 mg/mL to 1 mg/mL.
  12. 12 . The method according to claim 11 , wherein the concentration of the ovotransferrin in the chicken serum-free medium is 0.5 mg/mL.

Description

CROSS REFERENCE TO THE RELATED APPLICATIONS This application is a continuation application of International Application No. PCT/CN2024/130240, filed on Nov. 6, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD The present disclosure belongs to the field of biotechnologies, and specifically relates to a chicken serum-free medium for promoting proliferation of chicken primordial germ cells (PGCs) and a use of the chicken serum-free medium. BACKGROUND In the field of genetic breeding, PGCs have become important cells for gene editing and genetic improvement due to their unique developmental potential and genetic characteristics. PGCs originate from the ectoderm, migrate to the genital ridge at the early stage of embryonic development, and differentiate into female or male gametes with the development of a sexual gland. These characteristics make PGCs a key cell type for modifying genotypes from the source to prepare gene-edited animals. However, the in vitro culture and proliferation of PGCs has always been a technical challenge. The traditional culture methods are often faced with problems such as low cell proliferation efficiency and low cell survival rate, which seriously limit the application of PGCs in genetic breeding. Therefore, it is of great scientific significance and application value to explore novel culture systems and methods to improve the in vitro proliferation efficiency of PGCs. SUMMARY Technical problem to be solved: In view of the above-mentioned technical problems, the present disclosure provides a chicken serum-free medium for promoting proliferation of chicken PGCs and a use of the chicken serum-free medium. Ovotransferrin is a main component of chicken serum. In addition, ovotransferrin is transferrin widely present in organisms, and can promote the growth and proliferation of cells. During the culture of cells, ovotransferrin can provide the iron required by the growth of cells, and is involved in a variety of cellular metabolic processes, thereby contributing to the proliferation and differentiation of cells. Under specific culture conditions, ovotransferrin can significantly promote the in vitro proliferation of many types of cells. When an appropriate amount of ovotransferrin is added to a PGC medium, PGCs are in the optimal state, the number of PGCs increases significantly, and there is no obvious cell debris, indicating a significant improvement effect for the proliferation efficiency of PGCs. Technical solutions: In a first aspect, the present disclosure provides a chicken serum-free medium for promoting proliferation of chicken PGCs, where in the chicken serum-free medium, ovotransferrin is used instead of chicken serum. Preferably, the chicken serum-free medium further includes a Dulbecco's Modified Eagle Medium (DMEM), ultrafiltration water, and additives; and the additives include a concentrated calcium chloride stock solution, a B-27™ additive, a GlutaMax additive, an MEM non-essential amino acids solution, 2-mercaptoethanol, an EmbryoMax® nucleoside, sodium pyruvate, albumin, sodium heparin, basic fibroblast growth factor, activin A, and a penicillin-streptomycin mixed solution. Preferably, a concentration of the ovotransferrin in the chicken serum-free medium is 0.1 mg/mL to 1 mg/mL. Further, the concentration of the ovotransferrin in the chicken serum-free medium is 0.5 mg/mL. In a second aspect, the present disclosure provides a use of the chicken serum-free medium described in the first aspect in promoting proliferation of chicken PGCs. In a third aspect, the present disclosure provides a method for promoting proliferation of chicken PGCs, including the following steps: inoculating the chicken PGCs into the chicken serum-free medium described in the first aspect, and culturing at 37° C. for a specified period of time until a required number of cells are produced. Beneficial effects: The present disclosure discloses the use of ovotransferrin instead of chicken serum in a PGC culture system, and ovotransferrin can keep the self-renewing of PGCs to provide a basis for exploring a stable culture system. The chicken serum-free medium of the present disclosure can avoid the adverse effects caused by an unclear composition of chicken serum. In addition, the accurate use of various growth factors can provide excellent culture conditions and reduce the culture cost. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1C show the morphological observation results of PGCs cultured without chicken serum and with chicken serum in the present disclosure, where FIGS. 1A-1B show the influence of chicken serum-containing culture and chicken serum-free culture on the morphology of chicken PGCs, and FIG. 1C is a statistical chart of numbers of cells after 3 d of culture; FIGS. 2A-2F show the influence of ovotransferrin on the proliferation of PGCs in the present disclosure, where FIG. 2A shows the influence of estradiol and ovotransferrin at different concent