運用CRISPR/Cas9技術剔除海馬迴神經元的A型鉀離子通道

Abstract

A型鉀離子通道是一種電位閘型離子通道(Voltage-gated ion channel)，包含了電位閘型鉀離子通道KCNA4以及KCND家族。在過去的研究中指出，這些A型鉀離子通道，尤其是第四之二亞型(KCND2)，影響海馬迴CA1中的錐形細胞調控神經興奮性以及動作電位的向後傳導，而這些特性會促使海馬迴形成特別形式的神經可塑性。它們同樣在許多腦區中的記憶的穩固歷程、焦慮以及壓力行為上表現重要的腳色。常間回文重複序列叢集關聯蛋白(clustered, regularly interspaced, short palindromic repeats ,CRISPR/Cas9)，可以造成基因的插入/刪除突變(indel mutations)而使得特定基因剔除。這裡，我們使用第九血清型的腺病毒運送含有嚮導RNA的質體感染cre重組酶轉殖的鏈球菌Cas酵素之活體老鼠中的海馬迴神經元以剔除特定的KCND2基因，並且以腺病毒質體中帶有的紅螢光蛋白(mcherry)以及cre重組酶轉殖老鼠中帶有的綠螢光蛋白(GFP)來標定感染之細胞。再經過免疫染色確認剔除KCND2通道蛋白後，我們能夠利用這個技術進一步探討KCND2通道蛋白缺乏後的細胞電生理變化。

The A-type potassium channels, which involves the KCNA4 and the KCND family, are belongs to the voltage-gated ion channels. In previous studies, those A-type potassium channels, especially the KCND2, are important regulators of neuronal excitability and the action potential backpropagation which induce specific forms of synaptic plasticity in the CA1 pyramidal neurons of hippocampus. They also play essential roles in memory consolidation, anxiety and stress-related behaviors in many brain areas. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system can be used to knockout specific gene by inducing the insertion/deletion (indel) mutations. Herein, we delivered the single guide-RNAs (sgRNAs) using adeno-associated virus 9 (AAV9) vector to target the specific KCND2 (KV4.2) gene in the cre-dependent spCas9 mouse hippocampal neurons in vivo. I can identify the cells which were expressing sgRNAs and Cas9 proteins with reporter genes such as mcherry in AAV vectors as well as GFP in Cas9 heterozygous mice. After evaluating the knockout of KCND2 proteins by immunohistochemistry, we are going to use this techniques to investigate the protein depletion and electrophysiological changes of lacking of the KCND2 channels.