Hwang D Y, Carlezon W A, Isacson O, Kim K S
Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, MA 02478, USA.
Hum Gene Ther. 2001 Sep 20;12(14):1731-40. doi: 10.1089/104303401750476230.
Gene promoter systems that drive high-level, long-term, and cell-specific transgene expression are of great interest because of their potential utility for gene therapy. To generate an efficient promoter system specific for noradrenergic (NA) neurons, we multimerized an NA-specific cis-regulatory element (PRS2) identified in the human dopamine beta-hydroxylase (hDBH) promoter, and combined it with a minimal promoter (containing a TATA box and transcription start site). Forms of this synthetic promoter that contain 8 or more copies of PRS2 were >50 times more effective than the 1.15-kb hDBH promoter at driving reporter gene expression in cell lines originated from NA neurons. Neither the synthetic promoter nor the 1.15-kb hDBH promoter drove reporter gene expression in nonneuronal cells. Microinjections of an adenoviral vector containing the synthetic promoter directly into rat brain caused more strict NA-specific reporter gene expression than that caused by a vector containing the 1.15-kb hDBH promoter when the targeted region contained large numbers of NA neurons (locus coeruleus). Furthermore, the vector containing the synthetic promoter caused less nonspecific ("leaky") reporter gene expression than that caused by the vector containing the 1.15-kb hDBH promoter when the targeted region was devoid of NA neurons (cerebellum, dentate gyrus). Together, these studies provide in vitro and in vivo evidence that this novel synthetic promoter can target transgene expression to NA neurons even more efficiently and selectively than the naturally occurring, 1.15-kb hDBH promoter.
能够驱动高水平、长期且细胞特异性转基因表达的基因启动子系统因其在基因治疗中的潜在用途而备受关注。为了生成一种对去甲肾上腺素能（NA）神经元特异的高效启动子系统，我们将在人多巴胺β-羟化酶（hDBH）启动子中鉴定出的NA特异性顺式调控元件（PRS2）进行多聚化，并将其与一个最小启动子（包含TATA盒和转录起始位点）相结合。这种合成启动子中含有8个或更多PRS2拷贝的形式，在驱动源自NA神经元的细胞系中的报告基因表达方面，比1.15 kb的hDBH启动子有效50倍以上。合成启动子和1.15 kb的hDBH启动子均未在非神经元细胞中驱动报告基因表达。当将含有合成启动子的腺病毒载体直接显微注射到大鼠脑内时，在靶向区域含有大量NA神经元（蓝斑）的情况下，与含有1.15 kb hDBH启动子的载体相比，能引起更严格的NA特异性报告基因表达。此外，当靶向区域没有NA神经元（小脑、齿状回）时，含有合成启动子的载体引起的非特异性（“渗漏”）报告基因表达比含有1.15 kb hDBH启动子的载体少。总之，这些研究提供了体外和体内证据，表明这种新型合成启动子能够比天然存在的1.15 kb hDBH启动子更高效、更有选择性地将转基因表达靶向NA神经元。
