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A novel method to isolate primordial germ cells and its use for the generation of germline chimeras in chicken
Yasuhiro Yamamoto1, Fumitake Usui, Yoshiaki Nakamura
1Faculty of Agriculture, Shinshu University, Nagano 399-4598, Japan.
Insights
A new method efficiently isolates chick primordial germ cells (PGCs) from blood using ACK buffer, achieving high purity and enabling germline manipulation in chickens.
Area of Science:
- Poultry Science
- Developmental Biology
- Cell Biology
Background:
- Primordial germ cells (PGCs) are crucial for avian reproduction and germline transmission.
- Efficient isolation of PGCs is essential for genetic manipulation and research in chickens.
- Previous methods for PGC isolation were often complex and time-consuming.
Purpose of the Study:
- To develop a simple, rapid, and efficient method for isolating chick primordial germ cells (cPGCs) from embryonic blood.
- To assess the purity, recovery rate, and viability of cPGCs isolated using the novel method.
- To demonstrate the functional capacity of isolated cPGCs through in vitro culture and chimeric chicken production.
Main Methods:
- Isolation of cPGCs from circulating embryonic blood using ammonium chloride-potassium (ACK) buffer for red blood cell lysis.
- In vitro culture of cPGCs to further purify the cells and assess their viability and gene expression.
- Characterization of isolated cPGCs using germline-specific antibodies (e.g., anti-chicken vasa homolog [CVH]) and gene expression analysis (Cvh and Dazl).
- Production of chimeric chickens from cultured cPGCs and detection of donor cells in gonads.
Main Results:
- The ACK buffer method achieved a cPGC purity of 57.1% and a recovery rate of 90.3% after initial lysis, without affecting cPGC morphology.
- In vitro culture for 7 days increased PGC purity to 92.9%.
- Cultured cPGCs expressed germline markers (CVH) and genes (Cvh, Dazl).
- Chimeric chickens were successfully produced, with donor cPGCs detected in the gonads, confirming their biological function.
Conclusions:
- The novel ACK buffer-based method provides a simple and rapid approach for isolating chick PGCs from blood.
- This method significantly improves PGC purity and recovery, offering a more accessible tool for avian research.
- The successful production of chimeric chickens demonstrates the potential of this technique for germline manipulation in poultry.
Abstract:
A novel method was developed to isolate chick primordial germ cells (PGCs) from circulating embryonic blood. This is a very simple and rapid method for the isolation of circulating PGCs (cPGCs) using an ammonium chloride-potassium (ACK) buffer for lysis of the red blood cells. The PGCs were purified as in vitro culture proceeded. Most of the initial red blood cells were removed in the first step using the ACK lysis buffer. The purity of the cPGCs after ACK treatment was 57.1%, and the recovery rate of cPGCs from whole blood was 90.3%. The ACK process removed only red blood cells and it did not affect cPGC morphology. In the second step, the red blood cells disappeared as the culture progressed. At 7 days of in vitro culture, the purity of the PGCs was 92.9%. Most of these cells expressed germline-specific antibodies, such as those against chicken vasa homolog (CVH). The cultured PGCs expressed the Cvh and Dazl genes. Chimeric chickens were produced from these cultured PGCs, and the donor cells were detected in the gonads, suggesting that the PGCs had biological function. In conclusion, this novel isolation system for PGCs should be easier to use than previous methods. The results of the present study suggest that this novel method will become a powerful tool for germline manipulation in the chicken.
