Takeuchi Y, Yoshizaki G, Takeuchi T
Department of Aquatic Biosciences, Tokyo University of Fisheries, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
Mol Reprod Dev. 2001 Aug;59(4):380-9. doi: 10.1002/mrd.1044.
We describe a technique for producing germ-line chimeric rainbow trout, Oncorhynchus mykiss, by microinjection of the isolated blastomeres. FITC-labeled donor cells and non-labeled recipient embryos at various developmental stages between the early blastula and early gastrula stages were used for cell transplantation. The chimera formation rate and the degree of donor cell distribution in recipient embryos were evaluated at both the late gastrula stage (5 days post fertilization (dpf)) and the 40-somite stage (10 dpf). Among the six combinations of developmental stages of donor and recipient embryos, the combination of midblastula (2.5 dpf) donor cells and early blastula (1.5 dpf) recipient embryos gave the highest chimera formation rate and the best distribution pattern of donor cells. Using this combination, chimeric rainbow trout were produced with donor blastomeres from dominant orange-colored mutant embryos and wild-type recipient embryos. Of the 238 chimeric embryos produced, 28 (12%) hatched normally and 14 of the 28 fry (50%) had donor-derived orange body color. To test for germ-line transmission of donor cells, gametes obtained from the matured chimeras were fertilized with gametes from wild-type fish. Of the 19 matured chimeras, 6 (32%) yielded donor-derived orange-colored progeny, in addition to wild-type siblings. The contribution rates of donor cells in the germ-line ranged from 0.3 to 14%. This technique for producing germ-line chimeras should be a powerful tool for cell-mediated gene transfer in rainbow trout. Especially, if body color mutants are used for either donor cells or the host embryos, it will be possible to easily concentrate F1 transgenic embryos derived from transplanted donor cells by body color screening. Mol. Reprod. Dev. 59: 380-389, 2001.
我们描述了一种通过显微注射分离的卵裂球来制备生殖系嵌合虹鳟（Oncorhynchus mykiss）的技术。在囊胚早期和原肠胚早期之间的不同发育阶段，将异硫氰酸荧光素（FITC）标记的供体细胞和未标记的受体胚胎用于细胞移植。在原肠胚晚期（受精后5天（dpf））和40体节期（10 dpf）评估受体胚胎中的嵌合体形成率和供体细胞的分布程度。在供体和受体胚胎发育阶段的六种组合中，囊胚中期（2.5 dpf）供体细胞与囊胚早期（1.5 dpf）受体胚胎的组合产生了最高的嵌合体形成率和最佳的供体细胞分布模式。使用这种组合，用显性橙色突变胚胎的供体卵裂球和野生型受体胚胎产生了嵌合虹鳟。在产生的238个嵌合胚胎中，28个（12%）正常孵化，28条幼鱼中有14条（50%）具有供体来源的橙色体色。为了测试供体细胞的生殖系传递，从成熟的嵌合体获得的配子与野生型鱼的配子受精。在19个成熟的嵌合体中，6个（32%）除了产生野生型同胞外，还产生了供体来源的橙色后代个体。供体细胞在生殖系中的贡献率在0.3%至14%之间。这种制备生殖系嵌合体的技术应该是虹鳟细胞介导基因转移的有力工具。特别是，如果体色突变体用于供体细胞或宿主胚胎，将有可能通过体色筛选轻松富集来自移植供体细胞的F1转基因胚胎。《分子生殖与发育》59: 380 - 389, 2001年。
