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Isolation, culture and characterization of chicken primordial germ cells

Published online by Cambridge University Press:  20 March 2007

Tang Xin-Yan ,
Zeng Wei-Dong ,
Mi Yu-Ling ,
Liu Hong-Yun  and
Zhang Cai-Qiao
Tang Xin-Yan
Affiliation:
College of Animal Sciences, Zhejiang University, Hangzhou 310029, China
Zeng Wei-Dong
Affiliation:
College of Animal Sciences, Zhejiang University, Hangzhou 310029, China
Mi Yu-Ling
Affiliation:
College of Animal Sciences, Zhejiang University, Hangzhou 310029, China
Liu Hong-Yun
Affiliation:
College of Animal Sciences, Zhejiang University, Hangzhou 310029, China
Zhang Cai-Qiao*
Affiliation:
College of Animal Sciences, Zhejiang University, Hangzhou 310029, China
*
*Corresponding author. E-mail: [email protected]
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Abstract

Primordial germ cells (PGCs) were isolated from the genital ridges of chicken (Gallus domesticus) embryos at the 19th stage and purified by Ficoll density-gradient centrifugation. PGCs were co-cultured with somatic cells in preliminary culture and subcultured. Identification of PGCs was carried out by histochemical methods, including alkaline phosphatase (AKP) and periodic acid–Schiff (PAS). The proliferating activity of PGCs in subculture was demonstrated by immunocytochemistry of proliferating cell nuclear antigen (PCNA). Meanwhile, proliferating PGCs were compared under different culture conditions of 5–20% fetal cattle serum (FCS), insulin–transferrin–selenite (ITS) medium, conditioned medium (CM), 15% FCS+ITS, 15% FCS+40% CM. The results showed that the cultured PGCs were positive for AKP and PAS staining and displayed intensive proliferating activity by PCNA. The PGCs without centrifugation grew better than those with centrifugation. The PGCs formed larger colonies in media with 5% FCS or ITS than other media, indicating that 5% FCS or ITS supplemented media could be an ideal culture system for PGC proliferation in the PGC-somatic cell co-culture, in addition to the embryonic fibroblast feeder layer.

Keywords

cell proliferationchicken embryoin vitro cultureprimordial germ cell
Type
Research Article
Information
Chinese Journal of Agricultural Biotechnology , Volume 3 , Issue 3 , December 2006 , pp. 183 - 188
Copyright
China Agricultural University and Cambridge University Press 2006

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