利用CRISPR/Cas9技术构建CREB基因敲除细胞系并探讨CREB对APP基因表达的调控作用  

The Establishment of Stable CREB Gene Knock Out Cell Lines with CRISPR/Cas9 Technique and the Regulatory Effects of CREB on APP Gene Expression

作　　者：郭姗姗[1] 张冰莹 何文欣[1] 石晓光[1] 刘昆梅[1] 孙涛[1] 崔建奇[1,2] Guo Shanshan Zhang Bingying He Wenxin Shi Xiaoguang Liu Kunmei Sun Tao Cui Jianqi(Ningxia Key Laboratory of Cerebrocranial Diseases, Incubation Base of National Key Laboratory, Ningxia Medical University, Yinchuan 750004, China Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, China)

机构地区：[1]宁夏医科大学宁夏颅脑疾病重点实验室,省部共建国家重点实验室培育基地,银川750004 [2]宁夏医科大学基础医学院生物化学与分子生物学系,银川750004

出　　处：《中国细胞生物学学报》2017年第9期1147-1155,共9页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:81260197);宁夏脑计划项目(批准号:2016BZ07);宁夏医科大学2015年优势学科群建设科研项目(批准号:XY201511)资助的课题~~

摘　　要：环磷腺苷效应元件结合蛋白(c AMP responsive element-binding protein,CREB)是重要的核转录因子,有研究表明,在阿尔茨海默病患者中,CREB的表达降低,但CREB对于淀粉样前体蛋白(amyloid precursor protein,APP)的作用机制尚不清楚。该研究通过设计sg RNA序列并合成相应的寡核苷酸,将其克隆到p X459质粒中,构建CREB基因敲除的CRISPR/Cas9二合一表达载体。将构建成功的载体转染到HT22细胞中,通过TA克隆测序和T7E1酶切分析来验证sg RNA的活性。将构建的载体转染到HT22细胞中,用嘌呤霉素进行药物筛选,构建稳定的CREB基因敲除的细胞系。CREB对APP基因表达的调控作用通过检测其在正常HT22细胞和CREB基因敲除的HT22细胞系中的表达情况来进行确认。结果显示,成功构建了CRISPR/Cas9技术对CREB基因进行敲除的二合一表达载体,所设计的sg RNA的插入序列和开放阅读框架完全正确,经过酶切分析和序列测定,所构建的载体与实验设计相一致。CREB基因敲除的HT22细胞系其敲除效率达到90%以上。通过Western blot分析检测CREB对APP表达的影响,结果发现,当CREB基因敲除后,APP表达增加,而过表达CREB时,则APP蛋白质表达水平下降。这提示,CREB对APP的表达有下调作用,具体的机制还将继续研究。cAMP responsive element-binding protein（CREB） is an important nucleic transcriptional factor. It had been reported that the expression of CREB was suppressed in the patients with Alzheimer＇s disease, and at the meantime, the expression of amyloid precursor protein（APP） was increased. The mechanisms for the effects of CREB on APP gene expression is still unclear. In the current study, the specific sg RNA sequences for CREB gene were designed and the corresponding oligonucleotide were synthesized. The sg RNA were cloned into the Bbs I sites of pX459 plasmid to construct the 2-in-1 plasmid of CRISPR/Cas9 for CREB gene knock-out. The constructed plasmid was transfected into HT22 cells to evaluate the activities of sg RNA with TA cloning sequencing and T7E1 endonuclease cutting. When CRISPR/Cas9 activities were confirmed, the constructed plasmids were transfected into HT22 cells and the transfected cells were cultivated under the suppression of puromycin. In this way the stable cell lines with CREB gene knock-out were established. The regulatory effects of CREB on APP gene expression were assessed with the Western blot assay to analyze the level of APP protein in the normal HT22 and CREB gene knockout cell lines. The results demonstrated that the constructed 2-in-1 plasmid with CRISPR/Cas9 techniques for CREB gene knock-out was successful. The insert sg RNA sequence and the open reading frame were correctly identified by the endonuclease cutting and the DNA sequencing analysis. The constructed plasmid was consistent with the experimental design. The established stable cell lines had high efficacy for CREB gene knock-out and the efficiency reached over 90%. The results of Western blot assay illustrated that the level of APP protein increased when CREB gene had been knocked out and reduced when CREB gene overexpressed. Our work indicated that CREB could down regulate APP gene expression and the detailed mechanism for this was still under investigation.

关 键 词：CRISPR/Cas9 CREB 基因敲除 APP基因表达 阿尔茨海默病 

分 类 号：R749.16[医药卫生—神经病学与精神病学]