Vollum, Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Mol Cell Neurosci. 1992 Apr;3(2):152-61. doi: 10.1016/1044-7431(92)90019-x.
Somatostatin (SS) is a neuropeptide synthesized in the mammalian brain. To identify the cis-acting elements responsible for neural-specific expression we produced transgenic mice by microinjecting a 15-kb fragment of the mouse SS gene that contains both exons, the intron, 12 kb of the 5' flanking region, and 2 kb of the 3' flanking region. As a reporter, a 30-bp oligonucleotide was inserted in the 5' untranslated region of the SS gene. Four pedigrees of transgenic mice with different chromosomal integrations were identified by Southern blots. In situ hybridizations revealed an unexpected expression of the transgene in the paraventricular, supraoptic, and retro-chiasmatic nuclei of the hypothalamus. Conversely, areas with high levels of endogenous SS mRNA, including the periventricular nucleus of the hypothalamus, reticular nucleus of the thalamus, and hippocampus were devoid of transgene-encoded SS expression. Immunocytochemistry with a pro-SS antibody labeled the identical ectopic hypothalamic and the normal somatostatinergic neurons. Double-staining immunocytochemistry showed colocalization of SS and vasopressin (AVP) in all groups of hypothalamic magnocellular neurons while there was no colocalization of SS and oxytocin. In dehydrated transgenic mice, hypothalamic AVP and transgenic SS mRNA increased 2.5- and 2.3-fold, respectively. Our results indicate that the 15-kb fragment of the SS gene does not provide all the necessary elements to activate its own transcription in somatostatinergic neurons. In addition, the expression of the transgene in vasopressinergic magnocellular neurons suggests that the SS gene fragment contains sequences that are recognized by transcriptional activating factors present in these cells.
生长抑素（SS）是哺乳动物大脑中合成的一种神经肽。为了鉴定负责神经特异性表达的顺式作用元件，我们通过微注射小鼠 SS 基因的 15kb 片段产生了转基因小鼠，该片段包含两个外显子、内含子、5'侧翼区的 12kb 和 3'侧翼区的 2kb。作为报告基因，将 30bp 的寡核苷酸插入 SS 基因的 5'非翻译区。通过 Southern 印迹鉴定了具有不同染色体整合的四个转基因小鼠品系。原位杂交显示转基因在下丘脑的室旁核、视上核和视交叉后核中表达。相反，包括下丘脑室旁核、丘脑网状核和海马在内的内源性 SS mRNA 水平较高的区域缺乏转基因编码的 SS 表达。用 pro-SS 抗体进行免疫细胞化学标记了相同的异位下丘脑和正常生长抑素能神经元。双重免疫细胞化学染色显示 SS 和血管加压素（AVP）在所有下丘脑大细胞神经元中均有共定位，而 SS 和催产素没有共定位。在脱水的转基因小鼠中，下丘脑 AVP 和转基因 SS mRNA 分别增加了 2.5 倍和 2.3 倍。我们的结果表明，SS 基因的 15kb 片段不能提供激活其自身在生长抑素能神经元中转录所需的所有元件。此外，转基因在血管加压素能大细胞神经元中的表达表明 SS 基因片段包含可被这些细胞中存在的转录激活因子识别的序列。
