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

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 germlinespecific 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

Poultry genetic resource conservation using primordial germ cells

“…In the future, further investigations of cell surface markers of PGCs in several avian species other than the chicken, Japanese quail, and common pheasant are anticipated. Since avian embryonic blood cells are roughly composed of a small number of PGCs and a huge number of erythrocytes, two approaches can be used to purify PGCs: one is separation of PGCs from erythrocytes by density gradient methods using Ficoll or Nycodenz [6, 35], and the other is erythrocyte lysis using an ammonium chloride-potassium buffer [43]. Among these, the Nycodenz gradient centrifugation method achieves both high recovery and purity rates of PGCs in the chicken and Japanese quail [35].…”

Germline Replacement by Transfer of Primordial Germ Cells into Partially Sterilized Embryos in the Chicken1

“…Briefly, PBS(À) containing 10% fetal bovine serum (FBS) was used as a buffer instead of KAv-1 medium [27]. Some concentrated PGCs were labeled with the fluorescent lipophilic carbocyanine dye PKH-26 (Zynaxis, Malvern, PA) according to the protocol of Yamamoto et al [28]. Chicken PGCs are easily distinguished from erythrocytes by their morphological characteristics, such as large size and the presence of many refractive granules in the cytoplasm observed using phase-contrast microscopy [29].…”

Possibility to Establish Chicken Stem Cell from Non-germline Tissue; Detection of Colony-forming Cells after Chicken Fibroblast Culture and Subsequent Stem Cell Characterization

“…developing embryos, which yield a prior feasibility in biotechnological industries compared with the mammals. To date, there has been an achievement in producing germline chimeras by germ cell transfer into blood stream (Naito et al, 1994;Park et al, 2003;Kim et al, 2005;Yamamoto et al, 2007), and in generating interspecies (Kang et al, 2008) and transgenic (Ono et al, 1998;Van de Lavoir et al, 2006) chimeras. However, there are difficulties in germ cell retrieval from developing embryos and in culturing of germ cells without losing their activity in vitro.…”