Submitted March 1, 2007
Returned for revision March 20, 2007
Accepted April 10, 2007
Gamete Biology
A Novel Method to Isolate Primordial Germ Cells and Its Use for the Generation of Germline Chimeras in Chicken
Yasuhiro Yamamoto ,
Fumitake Usui ,
Yoshiaki Nakamura ,
Yohei Ito ,
Takahiro Tagami ,
Keijiro Nirasawa ,
Yuko Matsubara ,
Tamao Ono ,
and
Hiroshi Kagami *
* To whom correspondence should be addressed. E-mail: kagami{at}shinshu-u.ac.jp.
Abstract
A novel method was developed to isolate chick primordial germ cells (PGCs) from circulating embryonic blood. The method was very simple and quick for the isolation ofcirculating PGCs (cPGCs) using an Ammonium Chloride Potassium (ACK) buffer which lysed the red blood cells. Furthermore, the PGCs were purified as the in vitro culture proceeded. The majority of the initial red blood cells were removed at the first step using the ACK lysing buffer. The purity of the cPGCs was 57.1% following the ACK treatment, 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 the morphology of the cPGCs. In the second step, red blood cells disappeared as the days in culture advanced. At 7 days of in vitro culture, the purity of the PGCs was raised to 92.9%. Most of these cells expressed germline-specific antibodies; chicken vasa homolog (Cvh). The cultured PGCs were shown to express 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 compared to previous methods. These results suggest that this novel method will become a powerful tool for germline manipulation in the chicken.
Key words:
Gamete Biology •
Developmental biology •
Early development •
Gene regulation
