Studies on the differentiation of pigment cells with use of transgenic medaka fish bearing the gene for mouse tyrosinase

携带小鼠酪氨酸酶基因的转基因鱼是通过将其显微注射到卵母细胞核中或通过电穿孔将其导入橙色变种青冈鱼(Oryzias Latipe)的受精卵中而产生的。我们成功地获得了41条皮肤色素沉着的转基因鱼，前一种方法获得了3条，后一种方法获得了38条。这些转基因鱼的棕色皮肤色素沉积被发现是由于“无色素性黑素载体”中的黑色素生成造成的，这种黑色素载体分布在该物种的橙色变体中。使用抗鼠酪氨酸酶抗体的免疫荧光揭示了鼠型酪氨酸酶在这些黑素载体中的明显分布。对转基因鱼皮肤的电子显微镜观察显示，转基因鱼的黑素小体具有许多大小不均的网格状内部结构，这与鱼类黑素小体常见的“多囊”结构不同，而且在黄色素和白质粒中几乎没有检测到黑色素沉积。所有这些发现都强烈地表明，在青竹中，转基因是以一种细胞类型特异的方式表达的。转基因鱼和橙色变异体的鱼杂交产生的后代具有野生型棕色和橙色的色素，比率约为1：1，这表明根据孟德尔定律传递了转基因基因。

Transgenic fish bearing the gene for mouse tyrosinase are produced by microinjecting it into the oocyte nucleus of or by introducing it by electroporation into the fertilized egg of an orange-colored variant of medaka (Oryzias latipes). We successfully obtain 41 transgenic fish exhibiting brown skin pigmentation, 3 fish by the former method and 38 by the latter. Brown skin pigmentation in these transgenic fish is found to result from melanogenesis in "amelanotic melanophores", which are distributing in an orange-colored variant of this species. immunofluorescence using the anti-mouse tyrosinase antibody discloses and apparent distribution of mouse-type tyrosinase in these melanophores. Electron microscopy on the skin of transgenic fish discloses that the melanophores are installed with numerous, unevenly sized melanosomes having a lattice-like internal structure, which is different from "multivesicular" ones common to fish melanophores, and that little melanin deposition is detectable in xanthophores and leucophores. All these findings strongly suggest that the gene transfected is expressed under a cell-type specific manner in medaka. Crossing between a transgenic fish and a fish from an orange-colored variant yields offsprings having wild-type brown and orange-colored pigmentation at an approximate rate of 1 : 1, suggesting transmission of the transfected gene according to the Mendelian law.