Survival and Proliferation of Refined Chicken Circulating Primordial Germ Cells Cultured In Vitro

In recent years, much attention has been paid to the manipulation of primordial germ cells (PGCs) to produce transgenic chicken. For this purpose, the in vitro proliferation of PGCs would make gene targeting possible, promoting the efficiency of transgenes. However, few researches have been carried out on this topic. In the present study, we tried to culture chicken PGCs outside the body. The PGCs were isolated from circulating blood of chicken embryos by the method of Ficoll density centrifugation, and labeled with PKH26 red fluorescent cell linker. The cells were refined by selection with a fine glass pipette and a micromanipulator in order to avoid the contamination of PGCs with red cells. The refined cells were then cultured for a given period. The PGCs proliferated when they were cultured on feeder cells derived from the germinal ridges of chicken embryos at stage 27. During a 5 day culture period, the highest rate of increased PGCs was about 51% for one of the two test groups. Although some kinds of growth factors were supplemented in the culture media, no synergistic effect of chicken stem cell factor (chSCF) with murine leukemia inhibitory factor (LIF) and human basic fibroblast growth factor (bFGF) on the proliferation of PGCs was found. This result implies that the LIF and bFGF influencing the proliferation of PGCs may not be conserved between mammals and birds.