Klarsfeld A, Bessereau J L, Salmon A M, Triller A, Babinet C, Changeux J P
Unité de Neurobiologie Moléculaire, URA CNRS D1284, Institut Pasteur, Paris, France.
EMBO J. 1991 Mar;10(3):625-32. doi: 10.1002/j.1460-2075.1991.tb07990.x.
We have obtained transgenic mice expressing nuclearly targeted beta-galactosidase (nls-beta-gal) under the control of a chicken acetylcholine receptor alpha-subunit promoter. The expression of the transgene was detected in early somites, starting before embryonic day 9.5. In 13-day embryos, the expression pattern of the transgene closely paralleled that of the endogenous mouse alpha-subunit gene, assessed by in situ hybridization. Our results illustrate, with single-cell resolution, the tissue specificity of this alpha-subunit promoter during embryogenesis. After birth, the overall beta-galactosidase activity rapidly decreased with age. However, in diaphragms of newborn animals, beta-galactosidase activity selectively persisted in nuclei underlying the motor endplates. The latter were revealed by an acetylcholinesterase stain. Nls-beta-gal was also visualized by indirect immunofluorescence, while endplates were labelled with fluorescent alpha-bungarotoxin. Confocal microscopy unambiguously identified the more intensely stained nuclei as synaptic 'fundamental nuclei', and allowed estimates of relative staining levels. Thus an 842 bp acetylcholine receptor gene promoter confers preferential synaptic expression to a reporter gene within myofibres in vivo.
我们获得了在鸡乙酰胆碱受体α亚基启动子控制下表达核靶向β-半乳糖苷酶（nls-β-gal）的转基因小鼠。在胚胎第9.5天之前，早期体节中就检测到了转基因的表达。通过原位杂交评估，在13天的胚胎中，转基因的表达模式与内源性小鼠α亚基基因的表达模式密切平行。我们的结果以单细胞分辨率说明了该α亚基启动子在胚胎发生过程中的组织特异性。出生后，总体β-半乳糖苷酶活性随年龄迅速下降。然而，在新生动物的膈肌中，β-半乳糖苷酶活性选择性地持续存在于运动终板下方的细胞核中。后者通过乙酰胆碱酯酶染色显示。nls-β-gal也通过间接免疫荧光可视化，而终板则用荧光α-银环蛇毒素标记。共聚焦显微镜明确地将染色更强烈的细胞核鉴定为突触“基本核”，并允许估计相对染色水平。因此，一个842 bp的乙酰胆碱受体基因启动子在体内赋予报告基因在肌纤维内优先的突触表达。
