Coelho Geovanna C Z, Yo Isaac S, Mira-López Tatiana M, Monzani Paulo S, Arashiro Dilberto R, Fujimoto Takafumi, Senhorini José A, Yasui George S
Institute of Bioscience, São Paulo State University, São Paulo, Brazil.
Int J Dev Biol. 2019;63(1-2):57-65. doi: 10.1387/ijdb.180348gc.
The transplantation of primordial germ cells (PGCs) is a valuable tool for gene-banking and reconstitution by means of a germline chimera. For this technology, studies regarding developmental stages and traceability of PGCs are necessary. The objective of this study was to develop a micromanipulation procedure for the future establishment of cryobanks of PGCs in migratory characins. Incubation temperatures were evaluated at 22 ° C, 26 ° C, and 30 ° C in order to synchronize developmental stages. The highest hatching rates and the lowest abnormality rate arose at 26° C, which was considered to be the best incubation temperature. Enzymatic removal of the chorion was determined to be best using 0.05% pronase, in which the embryos presented better survival rates. In order to visualize PGCs in vivo, artificial GFP-nos1 3'UTR mRNA was injected and the migration route was observed in vivo as PGCs were visualized firstly at the segmentation stage (6 to 13 somites). The number of GFP positive cells ranged from 8 to 20 per embryo (mean of 13.8; n = 5). After hatching, GFP-positive cells increased to 14 to 27 embryos (mean of 19.8; n = 5). Visualization of the GFP-positive cells was possible at 10 days post hatching, and at this stage, the cells were positioned in the yolk extension region. This is the first report on PGC visualization in vivo in Neotropical fish; the obtained data provide information on the identification and migration of PGCs. The information presented in this work brings new insights in gene banking in Neotropical species and subsequent reconstitution through a germinal germline chimera.
原始生殖细胞（PGCs）移植是一种通过种系嵌合体进行基因库构建和重建的重要工具。对于这项技术而言，关于PGCs发育阶段和可追溯性的研究是必要的。本研究的目的是开发一种显微操作程序，以便未来在洄游型脂鲤中建立PGCs冷冻库。为了同步发育阶段，对22℃、26℃和30℃的孵育温度进行了评估。26℃时孵化率最高且异常率最低，该温度被认为是最佳孵育温度。经测定，使用0.05%链霉蛋白酶酶解去除卵膜效果最佳，在此条件下胚胎的存活率更高。为了在体内观察PGCs，注射了人工绿色荧光蛋白 - nos1 3'UTR mRNA，并在体内观察其迁移路径，因为PGCs首先在体节期（6至13对体节）被观察到。每个胚胎中绿色荧光蛋白阳性细胞的数量在8至20个之间（平均为13.8；n = 5）。孵化后，绿色荧光蛋白阳性细胞增加到每个胚胎14至27个（平均为19.8；n = 5）。孵化后10天可以观察到绿色荧光蛋白阳性细胞，此时这些细胞位于卵黄延伸区域。这是关于新热带鱼体内PGCs可视化的首次报道；所获得的数据提供了有关PGCs识别和迁移的信息。这项工作所呈现的信息为新热带物种的基因库构建以及随后通过生殖系嵌合体进行重建带来了新的见解。
