Zebrafish primordial germ cell cultures derived from vasa::RFP transgenic embryos
Zebrafish primordial germ cell cultures derived from vasa::RFP transgenic embryos
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DOI：
10.1089/scd.2007.0178
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发表时间：
2008-06-01
影响因子：
4
通讯作者：
Collodi, Paul
中科院分区：
文献类型：
--
作者：
Fan, Lianchun;Moon, Jesung;Collodi, Paul
虽然胚胎生殖细胞（EG）介导的基因转移已经成功地在小鼠中超过十年，这种方法是有限的，在其他物种由于分离的生殖细胞谱系（PGCs）的早期胚胎的祖细胞的数量很少，缺乏信息的细胞在体外培养的要求。本研究建立了斑马鱼胚胎PGCs的体外培养方法。使用在PGC特异性vasa启动子的控制下表达红色荧光蛋白（RFP）的转基因胚胎，使得有可能通过荧光激活细胞分选（FACS）分离PGC的纯群体，并通过计数在不同培养基中产生的荧光PGC菌落的数量来优化培养条件。从26体节期胚胎开始的培养物含有最高百分比的PGCs，其在体外增殖以产生集落。研究了生长因子，包括Kit配体a和B（Kitlga和Kitlgb）以及基质细胞衍生因子1a和1 B（Sdf-1a和Sdf-1 B）对PGC增殖的影响。当通过转染的饲养细胞将重组Kitlga和Sdf-1b添加到培养基中时，实现了斑马鱼PGC的最佳体外生长和存活，导致PGC集落数量加倍。从RT-PCR和原位杂交分析的结果表明，PGCs保持在文化表达的Kita受体，即使受体的表达没有检测到PGCs分离的FACS直接从解离的胚胎。在最佳生长条件下，PGCs在培养物中持续增殖至少4个月。从斑马鱼建立长期PGC培养物的能力将使在该模型物种中进行生殖细胞分化和EG细胞多能性的体外研究成为可能，并且可能对细胞介导的基因转移方法的发展有价值。
Although embryonic germ (EG) cell-mediated gene transfer has been successful in the mouse for more than a decade, this approach is limited in other species due to the difficulty of isolating the small numbers of progenitors of germ cell lineage (PGCs) from early-stage embryos and the lack of information on the in vitro culture requirements of the cells. In this study, methods were established for the culture of PGCs obtained from zebrafish embryos. Transgenic embryos that express the red fluorescent protein (RFP) under the control of the PGC-specific vasa promoter were used, making it possible to isolate pure populations of PGCs by fluorescence-activated cell sorting (FACS) and to optimize the culture conditions by counting the number of fluorescent PGC colonies produced in different media. Cultures initiated from 26-somite-stage embryos contained the highest percentage of PGCs that proliferated in vitro to generate colonies. The effect of growth factors, including Kit ligand a and b (Kitlga and Kitlgb) and stromal cell-derived factor 1a and 1b (Sdf-1a and Sdf-1b), on PGC proliferation was studied. Optimal in vitro growth and survival of the zebrafish PGCs was achieved when recombinant Kitlga and Sdf-1b were added to the culture medium through transfected feeder cells, resulting in a doubling of the number of PGC colonies. Results from RT-PCR and in situ hybridization analysis demonstrated that PGCs maintained in culture expressed the kita receptor, even though receptor expression was not detected in PGCs isolated by FACS directly from dissociated embryos. In optimal growth conditions, the PGCs continued to proliferate for at least 4 months in culture. The capacity to establish long-term PGC cultures from zebrafish will make it possible to conduct in vitro studies of germ cell differentiation and EG cell pluripotency in this model species and may be valuable for the development of a cell-mediated gene transfer approach.