一种检测线粒体核酸酶靶向剪切活性的新方法

doi:10.13560/j.cnki.biotech.bull.1985.2015.12.012

生物技术通报 ›› 2015, Vol. 31 ›› Issue (12): 81-90.doi: 10.13560/j.cnki.biotech.bull.1985.2015.12.012

一种检测线粒体核酸酶靶向剪切活性的新方法

魏迪^1,2,高敬^1,2,池振奋³,张癸荣^1,2,聂凌云^1,2

1.河北北方学院，张家口 075000；2.中国人民解放军总后勤部卫生部药品仪器检验所，北京 100071；3.中国科学院北京基因组研究所，北京 100101

收稿日期:2015-03-18 出版日期:2015-12-19 发布日期:2015-12-19
作者简介:魏迪，女，硕士研究生，研究方向:遗传学研究新技术与新方法；E-mail:18311012326@163.com
基金资助:
国家自然科学基金面上项目(31371346)

A Novel Method for Detecting Activity of Mitochondria-targeted Nuclease

Wei Di^1,2, Gao Jing^1,2, Chi Zhenfen³, Zhang Guirong^1,2, Nie Lingyun^1,2

1. Hebei North University，Zhangjiakou 075000；2. Institute for Drug and Instrument Control，Health Department，the General Logistics Department of People's Liberation Army，Beijing 100071；3. Beijing Institute of Genomics，Chinese Academy of Sciences，Beijing 100101

Received:2015-03-18 Published:2015-12-19 Online:2015-12-19

摘要/Abstract

摘要： 旨在建立一种简便检测线粒体DNA(mtDNA)核酸酶靶向剪切活性的方法。利用转基因技术，将一段含有两个靶向目标序列(T1、T2)的线粒体DNA序列随机整合到宿主基因组中，通过实时荧光定量PCR筛选单拷贝或低拷贝的单克隆转基因细胞株。将含有T1、T2的CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9质粒分别瞬时转染到所选细胞株中，靶向剪切核基因组，在靶向目标序列处造成DNA双链断裂，引发非同源末端连接修复机制，引入插入或缺失突变。观察测序峰图，证明两个靶向目标序列T1、T2均有剪切效率，且T1高于T2。建立了一种高效快速检测线粒体核酸酶靶向剪切活性的新方法。

关键词: 线粒体核酸酶, 转基因细胞株, 靶向修饰, CRISPR/Cas9, 实时荧光定量PCR

Abstract: Mitochondrial DNA(mtDNA)mutation has been associated with human mitochondrial disease. The precise correction of mutated mtDNA is considered an important strategy of mitochondrial therapy. Due to the lack of the complete repair system of DNA damage, a convenient method for detecting activity of mitochondria-targeted nuclease needs to be innovated urgently. Using transgenic technology, a mitochondrial DNA containing two target sequences(T1 and T2)was integrated into host genome randomly. Single or low copy monoclonal transgenic cell lines were selected by real-time fluorescence quantitative PCR. The two CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9 plasmids, which contained T1 and T2 target sequences, were transiently transfected into selected copy monoclonal transgenic cell lines. Target DNA double-strand breaks were caused by CRISPR/Cas9, followed by DNA insertion and deletion mutation via non-homologous end joining repair pathway. The cutting activities of two target sequences were proved by DNA sequencing peaks diagram, it was proved that two target sequences of T1 and T2 had the cutting activity, furthermore, T1 was better than T2. A novel method for rapidly and efficiently detecting activity of mitochondria-targeted nuclease is established.

Key words: mitochondria-targeted nuclease, transgenic cell line, targeted modification, CRISPR/Cas9, real-time fluorescence quantitative PCR

魏迪,高敬,池振奋,张癸荣,聂凌云. 一种检测线粒体核酸酶靶向剪切活性的新方法[J]. 生物技术通报, 2015, 31(12): 81-90.

Wei Di, Gao Jing, Chi Zhenfen, Zhang Guirong, Nie Lingyun. A Novel Method for Detecting Activity of Mitochondria-targeted Nuclease[J]. Biotechnology Bulletin, 2015, 31(12): 81-90.

参考文献

[1] Perez EE, Wang J, Miller JC, et al. Establishment of HIV-1 resistance in CD4⁺ T cells by genome editing using zinc-finger nucleases[J]. Nature Biotechnology, 2008, 26(7):808-816.
[2] Urnov FD, Miller JC, Lee YL, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases[J]. Nature, 2005, 435(7042):646-651.
[3] Baker M. Gene-editing nucleases[J]. Nature Methods, 2012, 9(1):23-26.
[4] Sternberg SH, Redding S, Jinek M, et al. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9[J]. Nature, 2014, 507(7490):62-67.
[5] Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9[J]. Science, 2014, 346(6213):1258096.
[6] Pozzan T, Magalhaes P, Rizzuto R. The comeback of mitochondria to calcium signalling[J]. Cell Calcium, 2000, 28(5-6):279-283.
[7] Starkov AA. The role of mitochondria in reactive oxygen species metabolism and signaling[J]. Annals of the New York Academy of Sciences, 2008, 1147:37-1152.
[8] Anderson S, Bankier AT, Barrell BG, et al. Sequence and organization of the human mitochondrial genome[J]. Nature, 1981, 290(5806):457-465.
[9] Schapira AH. Mitochondrial disease[J]. Lancet, 2006, 368(9529):70-82.
[10] Obara-Moszynska M, Maceluch J, Bobkowski W, et al. A novel mitochondrial DNA deletion in a patient with Kearns-Sayre syndrome:a late-onset of the fatal cardiac conduction deficit and cardiomyopathy accompanying long-term rGH treatment[J]. BMC Pediatrics, 2013, 13:27.
[11] Minczuk M, Papworth MA, Kolasinska P, et al. Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(52):19689-19694.
[12] Minczuk M, Papworth MA, Miller JC, et al. Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA[J]. Nucleic Acids Research, 2008, 36(12):3926-3938.
[13] Bacman SR, Williams SL, Pinto M, et al. Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs[J]. Nature Medicine, 2013, 19(9):1111-1113.
[14] Croteau DL, Stierum RH, Bohr VA. Mitochondrial DNA repair pathways[J]. Mutation Research, 1999, 434(3):137-148.
[15] Liu P, Demple B. DNA repair in mammalian mitochondria:Much more than we thought?[J]. Environmental and Molecular Mutagenesis, 2010, 51(5):417-426.
[16] Boesch P, Weber-Lotfi F, Ibrahim N, et al. DNA repair in organelles:Pathways, organization, regulation, relevance in disease and aging[J]. Biochim Biophys Acta, 2011, 1813(1):186-200.
[17] Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6096):816-821.
[18] Xue W, Chen S, Yin H, et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver[J]. Nature, 2014, 514(7522):380-384.
[19] Hu W, Kaminski R, Yang F, et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(31):11461-11466.
[20] Long C, McAnally JR, Shelton JM, et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA[J]. Science, 2014, 345(6201):1184-1188.
[21] O'Connell MR, Oakes BL, Sternberg SH, et al. Programmable RNA recognition and cleavage by CRISPR/Cas9[J]. Nature, 2014, 516(7530):263-266.
[22] Schon EA, DiMauro S, Hirano M. Human mitochondrial DNA:roles of inherited and somatic mutations[J]. Nature Reviews Genetics, 2012, 13(12):878-890.
[23] Yang L, Ding J, Zhang C, et al. Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR[J]. Plant Cell Reports, 2005, 23(10-11):759-763.
[24] Ingham DJ, Beer S, Money S, et al. Quantitative real-time PCR assay for determining transgene copy number in transformed plants[J]. Biotechniques, 2001, 31(1):132-134, 6-40.
[25] Song P, Cai C, Skokut M, et al. Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKERS^TM-derived transgenic maize[J], Plant Cell Reports, 2002, 20(10):948-954.
[26] 王晓建, 杨旭, 宋晓东, 等. 实时荧光定量PCR法检测转基因小鼠拷贝数[J]. 中国实验动物学报, 2007(3):170-174.
[27] Liddell L, Manthey G, Pannunzio N, et al. Quantitation and analysis of the formation of HO-endonuclease stimulated chromosomal translocations by single-strand annealing in Saccharomyces cerevisiae[J]. Journal of Visualized Experiments, 2011(55):e3150.

一种检测线粒体核酸酶靶向剪切活性的新方法

A Novel Method for Detecting Activity of Mitochondria-targeted Nuclease

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	陈小玲, 廖东庆, 黄尚飞, 陈英, 芦志龙, 陈东. 利用CRISPR/Cas9系统改造酿酒酵母的研究进展[J]. 生物技术通报, 2023, 39(8): 148-158.
[2]	杨玉梅, 张坤晓. 应用CRISPR/Cas9技术建立ERK激酶相分离荧光探针定点整合的稳定细胞株[J]. 生物技术通报, 2023, 39(8): 159-164.
[3]	余慧, 王静, 梁昕昕, 辛亚平, 周军, 赵会君. 宁夏枸杞铁镉响应基因的筛选及其功能验证[J]. 生物技术通报, 2023, 39(7): 195-205.
[4]	施炜涛, 姚春鹏, 魏文康, 王蕾, 房元杰, 仝钰洁, 马晓姣, 蒋文, 张晓爱, 邵伟. 利用CRISPR/Cas9技术构建MDH2敲除细胞株及抗呕吐毒素效应研究[J]. 生物技术通报, 2023, 39(7): 307-315.
[5]	刘晓燕, 祝振亮, 史广宇, 华梓宇, 杨晨, 张涌, 刘军. 乳腺生物反应器的表达优化策略[J]. 生物技术通报, 2023, 39(5): 77-91.
[6]	姚姿婷, 曹雪颖, 肖雪, 李瑞芳, 韦小妹, 邹承武, 朱桂宁. 火龙果溃疡病菌实时荧光定量PCR内参基因的筛选[J]. 生物技术通报, 2023, 39(5): 92-102.
[7]	宋海娜, 吴心桐, 杨鲁豫, 耿喜宁, 张华敏, 宋小龙. 葱鳞葡萄孢菌诱导下韭菜RT-qPCR内参基因的筛选和验证[J]. 生物技术通报, 2023, 39(3): 101-115.
[8]	穆德添, 万凌云, 章瑶, 韦树根, 陆英, 付金娥, 田艺, 潘丽梅, 唐其. 钩藤管家基因筛选及生物碱合成相关基因的表达分析[J]. 生物技术通报, 2023, 39(2): 126-138.
[9]	程静雯, 曹磊, 张艳敏, 叶倩, 陈敏, 谭文松, 赵亮. CHO细胞多基因工程改造策略的建立及应用[J]. 生物技术通报, 2023, 39(2): 283-291.
[10]	黄文莉, 李香香, 周炆婷, 罗莎, 姚维嘉, 马杰, 张芬, 沈钰森, 顾宏辉, 王建升, 孙勃. 利用CRISPR/Cas9技术靶向编辑青花菜BoZDS[J]. 生物技术通报, 2023, 39(2): 80-87.
[11]	王兵, 赵会纳, 余婧, 陈杰, 骆梅, 雷波. 利用CRISPR/Cas9系统研究REVOLUTA参与烟草叶芽发育的调控[J]. 生物技术通报, 2023, 39(10): 197-208.
[12]	李双喜, 华进联. 抗猪繁殖与呼吸障碍综合征基因编辑猪研究进展[J]. 生物技术通报, 2023, 39(10): 50-57.
[13]	林蓉, 郑月萍, 徐雪珍, 李丹丹, 郑志富. 拟南芥ACOL8基因在乙烯合成与响应中的功能分析[J]. 生物技术通报, 2023, 39(1): 157-165.
[14]	曹英芳, 赵新, 刘双, 李瑞环, 刘娜, 徐石勇, 高芳瑞, 马卉, 兰青阔, 檀建新, 王永. 抗除草剂大豆GE-J12实时荧光定量PCR检测方法的建立[J]. 生物技术通报, 2022, 38(7): 146-152.
[15]	刘静静, 刘晓蕊, 李琳, 王盈, 杨海元, 戴一凡. 利用CRISPR/Cas9技术建立OXTR基因敲除猪胎儿成纤维细胞系[J]. 生物技术通报, 2022, 38(6): 272-278.