摘要
研究一种蛋白质在神经元中的功能,最有效的方法之一是在该基因敲除动物的神经元中确认其表型.传统的用胚胎干细胞建立基因敲除动物模型的方法虽然稳定,但是复杂、耗时.近几年来,一种新型基因组编辑技术——CRISPR/Cas9,能够在不分裂的神经元中高效特异地敲除目的基因.本文研究了用CRISPR/Cas9系统敲除突触结合蛋白Ⅰ(synaptotagminⅠ,Syt1)基因后的小鼠海马培养神经元的电生理学特性.我们设计并构建了Syt1单导向RNA(Syt1 sgRNA)的慢病毒载体质粒,并用编码Cas9和Syt1 sgRNA的慢病毒感染培养的小鼠海马神经元,急性敲除神经元中Syt1基因(Syt1 sgRNA组),并用不靶向任何基因的Scramble sgRNA感染神经元作为阴性对照(Scramble组).通过全细胞膜片钳的方法检测单动作电位诱发的兴奋性突触后电流(single AP-eEPSC)、微小兴奋性突触后电流(mEPSCs)、高糖反应测量的即刻可释放囊泡池(RRP)以及10 Hz串刺激测量的囊泡释放概率(P_r).结果显示,Syt1 sgRNA组神经元丧失了Syt1的功能,并且与Syt1敲除(Syt1 KO)小鼠神经元的突触传递表型相似,而Scramble组神经元的各参数和野生型(WT)小鼠神经元相比没有显著性差异.本文为CRISPR/Cas9技术应用于神经元中基因的急性修饰提供了依据.
One of the effective methods to study the functions of a protein in neurons is to identify the phenotypes of cultured neurons from animals with genes knocked out. Traditional methods which use embryonic stem cells to establish gene knock-out animal models are stable, but complicated and time-consuming. Recently, the new genome editing technology, CRISPR/Cas9, can specifically and efficiently delete target genes in post-mitotic neurons. Here we investigated the electrophysiological phenotypes of the cultured mouse hippocampal neurons with the synaptotagmin I （Sytl） gene deleted by lentiviral introduction of CRISPR/Cas9. Sytl single guide RNA （sgRNA） was designed and constructed into the IentiCRISPR vector. Mouse hippocampal neurons are infected with lentivirus encoding Cas9 and Sytl sgRNA to acutely delete the Sytl gene （Sytl sgRNA group）. And Scramble sgRNA, which has no gene target, was used as a negative control （Scramble group）. Whole-cell patch-clamp recordings were used to test single action-potential evoked excitatory postsynaptic current （single AP-eEPSC）, miniature excitatory postsynaptic currents （mEPSCs）, sucrose responses measured readily releasable pool （RRP） and 10 Hz train stimulation measured release probability （P~）. Our results indicated that the Sytl sgRNA group showed loss of function of Sytl, and had the similar synaptic transmission phenotypes compared to the neurons of Sytl knock-out （KO） mice; and all the parameters tested had no significance between Scramble group neurons and wild-type （WT） neurons. This study provided evidence for the application of CRISPR/Cas9 technology in acute gene modification in neurons.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2017年第6期515-522,共8页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金面上项目(31471020)资助~~
