Yamamoto Yasuhiro, Usui Fumitake, Nakamura Yoshiaki, Ito Yohei, Tagami Takahiro, Nirasawa Keijiro, Matsubara Yuko, Ono Tamao, Kagami Hiroshi
Faculty of Agriculture, Shinshu University, Nagano 399-4598, Japan.
Biol Reprod. 2007 Jul;77(1):115-9. doi: 10.1095/biolreprod.107.061200. Epub 2007 Apr 18.
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.
开发了一种从循环胚胎血液中分离鸡原始生殖细胞（PGC）的新方法。这是一种非常简单快速的使用氯化铵-钾（ACK）缓冲液裂解红细胞来分离循环PGC（cPGC）的方法。随着体外培养的进行，PGC被纯化。在第一步中使用ACK裂解缓冲液去除了大部分初始红细胞。ACK处理后cPGC的纯度为57.１%，全血中cPGC的回收率为90.3%。ACK处理仅去除红细胞，不影响cPGC的形态。在第二步中，随着培养的进行红细胞消失。体外培养7天时，PGC的纯度为92.9%。这些细胞中的大多数表达种系特异性抗体，如抗鸡vasa同源物（CVH）的抗体。培养的PGC表达Cvh和Dazl基因。用这些培养的PGC产生了嵌合体鸡，并且在性腺中检测到了供体细胞，这表明PGC具有生物学功能。总之，这种用于PGC的新型分离系统应该比以前的方法更容易使用。本研究结果表明，这种新方法将成为鸡种系操作的有力工具。
