Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells

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  • Miyahara Daichi
    Faculty of Agriculture, Shinshu University, Japan Animal Breeding and Reproduction Research Division, NARO Institute of Livestock and Grassland Science, Japan
  • Mori Takafumi
    Faculty of Agriculture, Shinshu University, Japan
  • Makino Ryuichi
    Faculty of Agriculture, Shinshu University, Japan
  • Nakamura Yoshiaki
    Division of Germ Cell Biology, National Institute for Basic Biology, National Institute of Natural Sciences, Japan
  • Oishi Isao
    Health Research Institute, National Institute of Advance Industrial Science and Technology, Japan
  • Ono Tamao
    Faculty of Agriculture, Shinshu University, Japan
  • Nirasawa Keijiro
    Animal Breeding and Reproduction Research Division, NARO Institute of Livestock and Grassland Science, Japan
  • Tagami Takahiro
    Animal Breeding and Reproduction Research Division, NARO Institute of Livestock and Grassland Science, Japan
  • Kagami Hiroshi
    Faculty of Agriculture, Shinshu University, Japan

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Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enabling chicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containing PGCs obtained from 2.5-day-old chicken embryos according to the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced during culture in the basic medium containing either basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defining a culture condition enabling PGC-LCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline.

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