MOLECULAR REPRODUCTION AND DEVELOPMENT 75:274–281 (2008) Production of Quail (Coturnix japonica) Germline Chimeras Derived From In Vitro-Cultured Gonadal Primordial Germ Cells TAE SUB PARK, 1 MI A. KIM, 2 JEONG MOOK LIM, 1 AND JAE YONG HAN 1 * 1 Division of Animal Genetic Engineering, Department of Food and Animal Biotechnology, Seoul National University, Seoul, Korea 2 Avicore Biotechnology Institute, Inc., Gyeonggi-do, Korea ABSTRACT A previous report from our labo- ratory documented successful production of quail (Co- turnix japonica) germline chimeras by transfer of gonadal primordial germ cells (gPGCs). Subsequently, this study was designed to evaluate whether gPGCs can be maintained in vitro for extended period, and furthermore, these cultured PGCs can induce germline transmission after transfer into recipient embryos. In experiment 1, gonadal cells from the two strains (wild-type plumage (WP) and black (D) quail) were cultured in vitro for 10 days. Using antibody QCR1, we detected a continuous, significant (P ¼ 0.0002) increase in the number of WP, but not D, PGCs. QCR1-positive WP colonies began to form after 7 days in culture. On Day 10 of culture, 803 WP PGCs were present as a result of a continuous increase, whereas no D PGC colonies could be detected and the D gonadal stroma cells were rolled up. Differences in the PGCs or the gonadal stroma cells of the two different strains might account for these differ- ences. In experiment 2, WP PGC colonies were maintained in vitro up to Day 20 of culture, and 10- or 20-day-cultured PGCs were microinjected into dorsal aortas of 181 recipient D embryos. Thirty-five (19.3%) of the transplanted embryos hatched after incubation, and 25 (71.4%) of the hatchlings reached sexual maturity. Testcrossing of the sexually mature hatchlings resulted in three (10 days, 33.3%) and eight (20 days, 50.0%) germline chimeras respectively. This report is the first to describe successful production of germline chimera by transfer of in vitro-cultured gPGCs in quail. Mol. Reprod. Dev. 75: 274–281, 2008. ß 2007 Wiley-Liss, Inc. Key Words: quail; primordial germ cells (PGCs); microinjection; germline chimera; in vitro culture INTRODUCTION The unique pattern of germ cell migration in birds yields great advantages in the production of transgenic bioreactors and disease animal models (Perry and Sang, 1993; Sang, 1994). Avian germ cells have become extremely useful tools for transgenic research, and germ-cell-mediated production of germline chimeras has been attempted. To date, researchers have been successful at producing chicken germline chimeras by transferring primordial germ cells (PGCs) retrieved from the chicken germinal crescent (Wentworth et al., 1989; Jeong et al., 1999), blood stream (Naito et al., 1994), and embryonic gonads (Chang et al., 1997; Park et al., 2003b) into chicken embryos of different ages. Embryonic germ (EG) cells have been established by subculture of gonadal PGCs (gPGCs), and EG cells transplanted into recipient embryos also induced germ- line transmission in their progeny (Park and Han, 2000; Park et al., 2003a). Furthermore, transgenic chickens recently were reported by transferring the circulating PGCs in embryonic blood vessel after in vitro trans- duction of green fluorescent protein (GFP) gene (van de Lavoir et al., 2006). We have been attempting to produce quail germline chimera by techniques similar to those described above for the chicken. Quail has a number of advantages over chicken (low maintenance cost, small body size, and short generation time) that expand its utility as a model animal. Quail embryos are of optimal size for easy manipulation during all stages of embryonic develop- ment. Recently, we reported the successful production of quail germline chimera by transfer of gPGCs immedi- ately after collection (Kim et al., 2005). Subsequently, we found that the capacity of gPGCs to induce germline transmission was not decreased by culturing for 3 days in vitro prior to transplantation. In the present report, we describe the successful production of quail germline chimera from PGCs cultured for extended periods. The randomized pilot experiment was designed to evaluate whether quail PGCs could form colonies upon extended culture and whether they could induce germline transmission when transplanted into embryos. The information thus obtained will contribute to the development of effective transgenic technologies for quail. ß 2007 WILEY-LISS, INC. *Correspondence to: Jae Yong Han, Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea. E-mail: jaehan@snu.ac.kr Received 8 June 2007; Accepted 11 July 2007 Published online 14 September 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20821