Park Tae Sub, Kim Mi A, Lim Jeong Mook, Han Jae Yong
Division of Animal Genetic Engineering, Department of Food and Animal Biotechnology, Seoul National University, Seoul, Korea.
Mol Reprod Dev. 2008 Feb;75(2):274-81. doi: 10.1002/mrd.20821.
A previous report from our laboratory documented successful production of quail (Coturnix 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 = 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 differences. 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.
我们实验室之前的一份报告记录了通过转移性腺原始生殖细胞（gPGCs）成功培育出鹌鹑（日本鹌鹑）种系嵌合体。随后，本研究旨在评估gPGCs能否在体外长时间维持，此外，这些培养的PGCs在转移到受体胚胎后能否诱导种系传递。在实验1中，将两种品系（野生型羽毛（WP）和黑色（D）鹌鹑）的性腺细胞在体外培养10天。使用抗体QCR1，我们检测到WP品系而非D品系的PGCs数量持续显著增加（P = 0.0002）。培养7天后开始形成QCR1阳性的WP集落。在培养第10天，由于持续增加，出现了803个WP PGC，而未检测到D品系的PGC集落，且D品系的性腺基质细胞卷曲。两种不同品系的PGCs或性腺基质细胞的差异可能解释了这些不同。在实验2中，WP PGC集落在体外维持培养至第20天，并将培养10天或20天的PGCs显微注射到181个受体D胚胎的背主动脉中。35个（19.3%）移植胚胎孵化，25个（71.4%）幼雏达到性成熟。对性成熟幼雏进行测交分别产生了3个（10天，33.3%）和8个（20天，50.0%）种系嵌合体。本报告首次描述了通过转移体外培养的鹌鹑gPGCs成功培育种系嵌合体。
