Babcock A M, Standing D, Bullshields K, Schwartz E, Paden C M, Poulsen D J
Department of Psychology, Montana State University, Bozeman, MT 59717, USA.
Mol Ther. 2005 Jun;11(6):899-905. doi: 10.1016/j.ymthe.2005.02.016.
Hippocampal alpha-Ca2+/calmodulin-dependent protein kinase II (alpha-CaMKII) has been implicated in spatial learning, neuronal plasticity, epilepsy, and cerebral ischemia. In the present study, an adeno-associated virus (AAV) vector was designed to express green fluorescent protein (GFP) from the CBA promoter and a small hairpin RNA targeting alpha-CaMKII (AAV-shCAM) driven from the U6 promoter. The AAV-shCAM or control vector was microinfused into the rat hippocampus and behavioral testing conducted 19-26 days following surgery. Expression of the marker gene and alpha-CaMKII was evaluated 31 days following AAV infusion. GFP expression was localized to the hippocampus and extended +/-2 mm rostral and caudal from the injection site. Hippocampal alpha-CaMKII was significantly reduced following AAV-shCAM treatment as demonstrated using immunohistochemical and Western analysis. This suppression of alpha-CaMKII was associated with changes in exploratory behavior (open field task) and impaired place learning (water maze task). These results demonstrate the efficacy of a viral-based delivered shRNA to produce gene suppression in a specific circuit of the brain.
海马体α-钙调蛋白依赖性蛋白激酶II（α-CaMKII）与空间学习、神经元可塑性、癫痫和脑缺血有关。在本研究中，设计了一种腺相关病毒（AAV）载体，用于从CBA启动子表达绿色荧光蛋白（GFP），并从U6启动子驱动靶向α-CaMKII的小发夹RNA（AAV-shCAM）。将AAV-shCAM或对照载体微量注入大鼠海马体，并在手术后19-26天进行行为测试。在AAV注入后31天评估标记基因和α-CaMKII的表达。GFP表达定位于海马体，并从注射部位向头侧和尾侧延伸±2毫米。使用免疫组织化学和蛋白质印迹分析表明，AAV-shCAM处理后海马体α-CaMKII显著降低。α-CaMKII的这种抑制与探索行为（旷场试验）的变化和位置学习受损（水迷宫试验）有关。这些结果证明了基于病毒递送的短发夹RNA在大脑特定回路中产生基因抑制的有效性。
