BACKGROUND:Androgen regulation and prostate-specific expression of targeted genes in transgenic mice can be controlled by a small DNA fragment of the probasin (PB) promoter (-426 to +28 base pairs, bp). Although the small PB fragment was sufficient to direct prostate-specific expression, the low levels of transgene expression suggested that important upstream regulatory sequences were missing.

METHODS:To enhance transgene expression, a large fragment of the PB promoter (LPB, -11,500 to +28 bp) was isolated, linked to the bacterial chloramphenicol acetyl transferase (CAT) gene, and microinjected into CD1 mouse oocytes to generate transgenic mouse lines.

RESULTS:As shown by the immunohistochemical studies, CAT gene expression was restricted to the prostatic epithelial cells in a tissue-specific manner. High levels of CAT gene expression were observed in two of the six LPB-CAT transgenic lines. In Line 1, developmental regulation of LPB-CAT was detected early, from 1 to 4 weeks of age, with the activity of CAT increasing from 3 to 40,936 dpm/min/mg protein. Upon sexual maturation and elevated serum androgen levels (7 weeks of age), a further 18-fold rise in CAT activity occurred. Hormone ablation by castration in mature mice dramatically reduced transgene expression, whereas treatment with androgens returned LPB-CAT expression to precastration levels. In contrast, treatment with glucocorticoids had no significant effect on CAT gene expression. Zinc treatment of the castrated animals also increased LPB-CAT expression three- to four-fold in two prostatic lobes.

CONCLUSIONS:This study demonstrates that important regulatory DNA sequences located in the LPB fragment contribute to tissue-specific expression and greatly increase levels of transgene expression induced by androgens and zinc.

译文

背景 : 转基因小鼠中靶向基因的雄激素调节和前列腺特异性表达可以由probasin (PB) 启动子的小DNA片段 (-426至 + 28个碱基对,bp) 控制。尽管小的PB片段足以指导前列腺特异性表达,但低水平的转基因表达表明重要的上游调控序列缺失。
方法 : 为了增强转基因表达,PB启动子的大片段 (LPB,-分离出11,500至28 bp),与细菌氯霉素乙酰转移酶 (CAT) 基因连接,并将其微注射到CD1小鼠卵母细胞中,以生成转基因小鼠品系。
结果 : 如免疫组织化学研究所示,CAT基因表达以组织特异性方式限制在前列腺上皮细胞中。在六个LPB-CAT转基因品系中的两个中观察到高水平的CAT基因表达。在第1行中,从1至4周龄早期检测到LPB-CAT的发育调节,CAT的活性从3 dpm/min/mg增加到40,936 dpm/min/mg蛋白。性成熟和血清雄激素水平升高 (7周龄) 后,CAT活性进一步增加了18倍。成熟小鼠中去势的激素消融显着降低了转基因表达,而雄激素治疗使LPB-CAT表达恢复到去势前水平。相反,糖皮质激素治疗对CAT基因表达没有显着影响。Cast割动物的锌治疗还使两个前列腺叶中的LPB-CAT表达增加了三到四倍。
结论 : 这项研究表明,位于LPB片段中的重要调控DNA序列有助于组织特异性表达,并大大提高了雄激素和锌诱导的转基因表达水平。

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