Santamaria Julie, Khalfallah Olfa, Sauty Claire, Brunet Isabelle, Sibieude Mathieu, Mallet Jacques, Berrard Sylvie, Lecomte Marie-José
Laboratoire de Génétique Moléculaire de la Neurotransmission et des Processus Neurodégénératifs, Centre National de la Recherche Scientifique UMR 7091, Hôpital de la Pitié-Salpétrière, Paris, France.
J Neurosci Res. 2009 Feb;87(2):532-44. doi: 10.1002/jnr.21866.
RNA interference (RNAi) is a potent mechanism for local silencing of gene expression and can be used to study loss-of-function phenotypes in mammalian cells. We used RNAi to knockdown specifically the expression of choline acetyltransferase (ChAT), the enzyme of acetylcholine biosynthesis, both in cultured cells and in the adult brain. We first identified a 19-nucleotide sequence in the coding region of rat and mouse ChAT transcripts that constitutes a target for potent silencing of ChAT expression by RNAi. We generated a lentiviral vector that produces both a small hairpin RNA (shRNA) targeting ChAT mRNAs and the enhanced green fluorescent protein (EGFP) reporter protein to facilitate identification of transduced cells. In the cholinergic cell line NG108-15, there was at least 90% less of the ChAT protein, as measured by assaying its enzymatic activity, 3 days postinfection with this vector than in cells infected with a control vector. The vector was used to transduce cholinergic neurons in vivo and reduced ChAT expression strongly and specifically in the cholinergic neurons of the medial septum in adult rats, without affecting the expression of the vesicular acetylcholine transporter. This lentiviral vector is thus a powerful tool for specific inactivation of cholinergic neurotransmission and can therefore be used to study the role of cholinergic nuclei in the brain. This lentiviral-mediated RNAi approach will also allow the development of new animal models of diseases in which cholinergic neurotransmission is specifically altered.
RNA干扰（RNAi）是一种使基因表达局部沉默的有效机制，可用于研究哺乳动物细胞中的功能缺失表型。我们利用RNAi在培养细胞和成年大脑中特异性敲低胆碱乙酰转移酶（ChAT）的表达，ChAT是乙酰胆碱生物合成的酶。我们首先在大鼠和小鼠ChAT转录本的编码区鉴定出一个19个核苷酸的序列，该序列构成了通过RNAi有效沉默ChAT表达的靶点。我们构建了一种慢病毒载体，它既能产生靶向ChAT mRNA的小发夹RNA（shRNA），又能产生增强型绿色荧光蛋白（EGFP）报告蛋白，以方便鉴定转导细胞。在胆碱能细胞系NG108-15中，用该载体感染3天后，通过检测其酶活性发现，ChAT蛋白含量比感染对照载体的细胞至少低90%。该载体用于体内转导胆碱能神经元，能强烈且特异性地降低成年大鼠内侧隔胆碱能神经元中的ChAT表达，而不影响囊泡乙酰胆碱转运体的表达。因此，这种慢病毒载体是特异性失活胆碱能神经传递的有力工具，可用于研究胆碱能核团在大脑中的作用。这种慢病毒介导的RNAi方法还将有助于开发胆碱能神经传递发生特异性改变的新型疾病动物模型。
