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A Novel Method to Isolate Primordial Germ Cells and Its Use for the Generation of Germline Chimeras in Chicken¹

Faculty of Agriculture,³ Shinshu University, Nagano 399-4598, Japan Animal Breeding and Reproduction Research Team,⁴ National Institute of Livestock and Grassland Science (NILGS), Ibaraki 305-0901, Japan Transgenic Animal Research Center,⁵ National Institution of Agrobiological Sciences (NIAS), Ibaraki 305-0901, Japan

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.

developmental biology, early development, gamete biology, gene regulation

¹Supported in part by Grants-in-aid from the Ministry of Education, Science, Sports and Culture of the Japanese Government (nos. 15380190, 15658081, and 18380165, to H.K.).

Correspondence: ²Hiroshi Kagami, Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4598, Japan. FAX: 81 265 77 1419; e-mail: kagami{at}shinshu-u.ac.jp