title: Improvement of transfection efficiency in cultured chicken primordial germ cells by percoll density gradient centrifugation.
abstract: Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifying the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.
authors.affiliation: Health Research Institute, National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka, Japan. oishi-i@aist.go.jp
authors.forename: Isao
authors.initials: I
authors.lastname: Oishi
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods*; D002642:Chick Embryo; D002645:Chickens; D004247:DNA / Q000235:genetics; D005854:Germ Cells / Q000166:cytology* / Q000378:metabolism* / Q000637:transplantation; D006066:Gonads / Q000166:cytology / Q000254:growth & development; D011205:Povidone; D051381:Rats; D012822:Silicon Dioxide; D014162:Transfection / Q000379:methods*
chemical_list: C016039:Percoll; D012822:Silicon Dioxide; D004247:DNA; D011205:Povidone