Belenky M, Castel M, Young W S, Gainer H, Cohen S
Department of Experimental Zoology, Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
Brain Res. 1992 Jun 26;583(1-2):279-86. doi: 10.1016/s0006-8993(10)80034-4.
Cell-specific expression of the rat oxytocin (OT)-neurophysin transgene in mice was achieved using a construct containing both OT and vasopressin genes (Young III, W.S., Reynolds, K., Shepard, E.A., Gainer, H. and Castel, M., Cell-specific expression of the rat oxytocin gene in transgenic mice, J. Neuroendocrinol., 2 (1990) 1-9). The present study describes the distribution of the protein products of these genes in various regions of the cell, and determines whether the transgenic rat and endogenous mouse OT-neurophysins are colocalized within the same neurosecretory granules. Two monoclonal antibodies against OT-neurophysins were used: PS38 which can react with both rat and mouse OT-neurophysin (pan-specific), and PS67 which is specific for rat OT-neurophysin only. Various approaches to double immunolabeling at the ultrastructural level were employed; these included: (1) pre-embedding immunoperoxidase followed by post-embedding immunogold; (2) post-embedding immunolabeling using gold particles of different sizes; and (3) labeling of consecutive ultrathin sections with different antibodies. Results from each of these approaches showed that both in the transgenic mouse and in the rat (used as control), immunocytochemical labeling for both PS38 and PS67 occurred in the same OT-ergic neurosecretory granules. In the control mouse, only PS38 elicited labeling. Hence, it may be concluded that the protein and peptide products of the transgene and the endogenous gene for OT-neurophysin are being processed similarly in the cell and finally concentrated together in the same neurosecretory granules.
利用一个同时包含催产素（OT）和加压素基因的构建体，实现了大鼠OT-神经垂体素转基因在小鼠中的细胞特异性表达（扬三世，W.S.，雷诺兹，K.，谢泼德，E.A.，盖纳，H.和卡斯特尔，M.，转基因小鼠中大鼠催产素基因的细胞特异性表达，《神经内分泌学杂志》，2（1990）1 - 9）。本研究描述了这些基因的蛋白质产物在细胞各个区域的分布，并确定转基因大鼠和内源性小鼠OT-神经垂体素是否共定位于同一神经分泌颗粒中。使用了两种针对OT-神经垂体素的单克隆抗体：PS38，它能与大鼠和小鼠的OT-神经垂体素都发生反应（泛特异性），以及仅对大鼠OT-神经垂体素特异的PS67。采用了多种在超微结构水平进行双重免疫标记的方法；这些方法包括：（1）包埋前免疫过氧化物酶法，随后进行包埋后免疫金法；（2）使用不同大小金颗粒的包埋后免疫标记法；以及（3）用不同抗体对连续超薄切片进行标记。这些方法中的每一种的结果都表明，在转基因小鼠和大鼠（用作对照）中，PS38和PS67的免疫细胞化学标记都出现在相同的OT能神经分泌颗粒中。在对照小鼠中，只有PS38引发标记。因此，可以得出结论，转基因和内源性OT-神经垂体素基因的蛋白质和肽产物在细胞中以相似的方式进行加工，最终共同浓缩在相同的神经分泌颗粒中。
