Establishment of the long-term in vitro culture system for chicken primordial germ cells

• Main Authors: Huang-Ting Lu, 盧奐婷
• Other Authors: Lih-Ren Chen
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/u5n4rd

碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 92 === 　　Embryonic germ cells derived from primordial germ cells (PGCs) are undifferentiated and pluripotent. The objective of this study was to establish the long-term in vitro culture system for chicken PGCs. Primitive gonads were collected from 5.5-day-old (stage 28) chicken embryos and dissociated in 0.05% trypsin solution supplemented with 0.53mM EDTA. After centrifugation at 200×g for 5 min, these cells were seeded onto 4-well plates which were pre-coated with 0.1% gelatin. Each of the four different conditioned medium [CEF (non-inactivation), CEF (inactivation), STO (non-inactivation), STO (inactivation)] supplemented with growth factors (mLIF, hSCF, hbFGF, hIGF-1, hIL-11) was used to maintain the growth of PGCs. The result showed that all the conditioned media could promote the growth and colony formation of PGCs in vitro, in particular the medium conditioned by CEF (inactivation). These PGC-derived colonies were positively stained with PAS (a PGC-specific stain) and anti-SSEA-1(a monoclonal antibody specific to undifferentiated embryonic stem cells). After transferring to the recipient embryos with EGFP-transfected cultured gPGCs, EGFP gene was detected in the gonads of recipients, and the migration of PGC was proved. These data indicate that the long-term in vitro culture system of chicken PGCs has been preliminarily established in this study. The characteristics and the differentiation capacity of these PGC-derived cells will be studied further.