Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2017, Vol. 37 Issue (7): 80-87    DOI: 10.13523/j.cb.20170714
技术与方法     
位点特异整合微环DNA的体内制备
聂永强1, 马海燕1,2, 马晴雯1,2
1. 上海交通大学附属儿童医院 上海市儿童医院 上海交通大学医学遗传研究所 上海 200040;
2. 卫生部医学胚胎分子生物学重点实验室暨上海市胚胎与生殖工程重点实验室 上海 200040
An in vivo Robust System for Generation of Site-specific Integration Minicircle DNA Vector
NIE Yong-qiang1, MA Hai-yan1,2, MA Qing-wen1,2
1. Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China;
2. Key Laboratory of Embryo Molecular Biology, Ministry of Health & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China
 全文: PDF(842 KB)   HTML
摘要: 目的:应用诱导表达的LR克隆酶系统,建立一种在细菌体内获得基于链霉菌噬菌体ФC31整合酶系统的位点特异整合型微环DNA的方法,为实现无细菌骨架等冗余序列的转基因奠定基础。方法:构建包含阿拉伯糖启动子的LR克隆酶系统和ФC31整合酶系统的亲本质粒,在L-阿拉伯糖的诱导下重组产生表达ФC31整合酶的微质粒和包含有目的基因和attB位点等元件的微环DNA。以限制性内切核酸酶酶切电泳定性其重组效率,qPCR定量分析微环、微质粒及亲本质粒的比例,定量计算重组效率。观察随着诱导时间的推进微环/微质粒值的变化。结果:细菌体内LR克隆酶系统可有效催化亲本质粒的重组,重组率达85%以上。相比商品化LR克隆酶体外反应具有更高的稳定性而且更经济。结论:获得了一种高效、稳定的细菌体内产生位点特异性整合型微环DNA的亲本质粒。
关键词: 微环DNA阿拉伯糖启动子ФC31整合酶LR克隆酶系统位点特异性整合    
Abstract: Objective: Transgenic technology has an important application value in the field of gene therapy, transgenic animals, genome modification and other biomedical researches. Many researches demonstrated that ФC31 integrase system was an efficient tool for site-specific integration, but the biggest drawback of this system is the integration of the bacterial backbone in the attB donor plasmid. The minicircle DNA is an expression vector containing only the target gene, preparation of minicircle DNA based on ФC31 integrase system is expected to avoid the introduction of bacterial backbone at the integration site, with improved biological safety of the system. The classic procedure for minicircle DNA preparation is laborious with low yield and unstable efficiency. In order to solve above problems, a feasible and effective method for obtaining site-specific integrated minicircle DNA by combining the LR clonase system and the Streptomyces phage ФC31 integrase system were established, and laying a foundation for the construction of transgenic cells without redundant sequences such as bacterial backbone. Methods: The parental plasmid containing LR clonase system and ФC31 integrase system is constructed, it can be recombined by L-arabinose induced LR clonase in bacteria to produce a miniplasmid expressing ФC31 integrase in eukaryotic cells and a minicircle DNA containing the original genes such as the target gene and attB site. The recombination efficiency was qualitatively analyzed by restriction endonuclease digestion and quantitatively analyzed by calculating the proportion of minicircle DNA, miniplasmid and the parental plasmid. The change of the ratio of minicircle DNA/miniplasmid was observed along with the induction time. Results: A validation plasmid and a parental plasmid for the production of site-specific integrated minicircle DNA were constructed.The LR clonase system could effectively catalyze the recombination of attL and attR in the parental plasmid in bacteria, and the recombination rate was more than 85%. Conclusion: A parental plasmid which can be recombined to generate site-specific integration minicircle DNA by L-arabinose induction in bacteria with higher and more stable efficiency when compare to the in vitro LR recombination were constructed, which will expand the application potential of minicircle DNA in transgenic researches.
Key words: LR clonase system    ФC31 integrase system    Minicircle DNA    Arabinose promoter    Site-specific integration
收稿日期: 2017-03-06 出版日期: 2017-07-25
ZTFLH:  Q78  
基金资助: 上海市卫生局青年基金资助项目(20154Y0152)
通讯作者: 马晴雯     E-mail: maqingwen@hotmail.com
服务  
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章  
聂永强
马海燕
马晴雯

引用本文:

聂永强, 马海燕, 马晴雯. 位点特异整合微环DNA的体内制备[J]. 中国生物工程杂志, 2017, 37(7): 80-87.

NIE Yong-qiang, MA Hai-yan, MA Qing-wen. An in vivo Robust System for Generation of Site-specific Integration Minicircle DNA Vector. China Biotechnology, 2017, 37(7): 80-87.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170714        https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I7/80

[1] Maniar L E G, Maniar J M, Chen Z, et al. Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level. Molecular Therapy, 2013, 21(1): 131-138.
[2] Gaspar V, de Melo-Diogo D, Costa E, et al. Minicircle DNA vectors for gene therapy: advances and applications. Expert Opin Biol Ther, 2015, 15(3): 353-379.
[3] Karow M, Calos M P. The therapeutic potential of PhiC31 integrase as a gene therapy system. Expert Opin Biol Ther, 2011, 11(10): 1287-1296.
[4] Chalberg T W, Portlock J L, Olivares E C, et al. Integration specificity of phage phiC31 integrase in the human genome. J Mol Biol, 2006, 357(1): 28-48.
[5] Bigger B W, Tolmachov O, Collombet J M, et al. An araC-controlled bacterial cre expression system to produce DNA minicircle vectors for nuclear and mitochondrial gene therapy. J Biol Chem, 2001, 276(25): 23018-23027.
[6] Jechlinger W, Azimpour T C, Lubitz W, et al. Minicircle DNA immobilized in bacterial ghosts: in vivo production of safe non-viral DNA delivery vehicles. J Mol Microbiol Biotechnol, 2004, 8(4): 222-231.
[7] Chen Z Y, He C Y, Ehrhardt A, et al. Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol Ther, 2003, 8(3): 495-500.
[8] Tasic B, Hippenmeyer S, Wang C, et al. Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc Natl Acad Sci USA, 2011, 108(19): 7902-7907.
[9] Landy A. Dynamic, structural, and regulatory aspects of iambda site-specific recombination. Annual Review of Biochemistry, 1989, 58(1): 913-941.
[10] Liang X, Peng L, Baek C, et al. Single step BP/LR combined gateway reactions. BioTechniques, 2013, 55(5): 265-268.
[11] 刘浏. 位点特异整合型微环DNA在转基因研究中的应用. 上海:上海交通大学, 医学院,2014. Liu L.The Application of Site-specific Integration Minicircle DNA in Transgenic Research.Shanghai:Shanghai Jiao Tong University, Medical School, 2014.
[12] Groth A C, Olivares E C, Thyagarajan B, et al. A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci USA, 2000, 97(11): 5995-6000.
[13] 刘浏, 周在威, 马晴雯. 位点特异整合型微环DNA的构建及应用. 中国医药生物技术, 2014,9(1): 13-19. Liu L, Zhou Z W, Ma Q W.An improved method to generate site-specific integration minicircle DNA. Chin Med Biotechnol, 2014, 9(1):13-19.
[14] Mayrhofer P, Schleef M, Jechlinger W. Use of minicircle plasmids for gene therapy. Methods Mol Biol, 2009, 542: 87-104.
[15] Darquet A M, Cameron B, Wils P, et al. A new DNA vehicle for nonviral gene delivery: supercoiled minicircle. Gene Ther, 1997, 4(12): 1341-1349.
[16] Nehlsen K, Broll S, Bode J. Replicating minicircles: Generation of nonviral episomes for the efficient modification of dividing cells-research article. Gene Therapy and Molecular Biology, 2006, 10B: 233-243.
[17] Kay M A, He C, Chen Z. A robust system for production of minicircle DNA vectors. Nature Biotechnology, 2010, 28(12): 1287-1296.
[18] Diamantino T, Pereira P, Queiroz J A, et al. Minicircle DNA purification using a CIM(R) DEAE-1 monolithic support. J Sep Sci, 2016, 39(18): 3544-3549.
[19] Chen Z Y, He C Y, Kay M A. Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo. Hum Gene Ther, 2005, 16(1): 126-131.
[20] Bi Y, Liu X, Zhang L, et al. Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by Streptomyces phage phiC31 integrase. BMC Mol Biol, 2013, 14: 20.
[21] Thyagarajan B, Olivares E C, Hollis R P, et al. Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Mol Cell Biol, 2001, 21(12): 3926-3934.
[22] Chalberg T W, Portlock J L, Olivares E C, et al. Integration specificity of phage phiC31 integrase in the human genome. J Mol Biol, 2006, 357(1): 28-48.
[23] Ortiz-Urda S, Thyagarajan B, Keene D R, et al. Stable nonviral genetic correction of inherited human skin disease. Nat Med, 2002, 8(10): 1166-1170.
[24] Quenneville S P, Chapdelaine P, Rousseau J, et al. Nucleofection of muscle-derived stem cells and myoblasts with phi C31 integrase: Stable expression of a full-length-dystrophin fusion gene by human myoblasts. Molecular Therapy, 2004, 10(4): 679-687.
[25] Jechlinger W. Optimization and delivery of plasmid DNA for vaccination. Expert Rev Vaccines, 2006, 5(6): 803-825.
[26] Pang A S D. Production of antibodies against Bacillus thuringiensis delta-endotoxin by injecting its plasmids. Biochemical and Biophysical Research Communications, 1994, 202(3): 1227-1234.
[27] Woodard L E, Hillman R T, Keravala A, et al. Effect of nuclear localization and hydrodynamic delivery-induced cell division on phiC31 integrase activity. Gene Ther, 2010, 17(2): 217-226.
[28] Ma B G, Duan X Y, Niu J X, et al. Expression of stilbene synthase gene in transgenic tomato using salicylic acid-inducible Cre/loxP recombination system with self-excision of selectable marker. Biotechnol Lett, 2009, 31(1): 163-169.
[1] 陈鑫洁,钱芷兰,刘启,赵清,张元兴,蔡孟浩. 毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸*[J]. 中国生物工程杂志, 2021, 41(10): 52-61.
[2] 王伟东,杜加茹,张运尚,樊剑鸣. CRISPR/Cas9在人病毒感染相关疾病治疗研究中的应用*[J]. 中国生物工程杂志, 2020, 40(12): 18-24.
[3] 徐应永. 基因治疗产品的开发现状与挑战[J]. 中国生物工程杂志, 2020, 40(12): 95-103.
[4] 王玥,牟彦双,刘忠华. 基于CRISPR/Cas系统的单碱基编辑技术研究进展*[J]. 中国生物工程杂志, 2020, 40(12): 58-66.
[5] 雷海英,赵青松,白凤麟,宋慧芳,王志军. 利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*[J]. 中国生物工程杂志, 2020, 40(12): 49-57.
[6] 薛瑞,姚林,王瑞,罗正山,徐虹,李莎. 重组贻贝足蛋白的研究进展与应用*[J]. 中国生物工程杂志, 2020, 40(11): 82-89.
[7] 陈庆宇,王鲜忠,张姣姣. 基因技术在治疗2型糖尿病中的应用*[J]. 中国生物工程杂志, 2020, 40(11): 73-81.
[8] 何秀娟,胡凤枝,刘秋丽,刘玉萍,祝玲,郑文云. 乳腺癌细胞QSOX1的CRISPR/Cas9基因编辑及其对增殖侵袭的影响研究*[J]. 中国生物工程杂志, 2020, 40(11): 1-9.
[9] 吕一凡,李更东,薛楠,吕国梁,时邵辉,王春生. LbCpf1基因的原核表达、纯化与体外切割检测 *[J]. 中国生物工程杂志, 2020, 40(8): 41-48.
[10] 张保惠,熊华龙,张天英,袁权. 基于水疱性口炎病毒(VSV)的溶瘤病毒研究进展 *[J]. 中国生物工程杂志, 2020, 40(6): 53-62.
[11] 武瑞君,李治非,张鑫,濮润,敖翼,孙燕荣. 新冠病毒抗体药物研发进展及展望分析[J]. 中国生物工程杂志, 2020, 40(5): 1-6.
[12] 刘迪,张洪春. 慢性阻塞性肺疾病基因工程动物模型研究进展 *[J]. 中国生物工程杂志, 2020, 40(4): 59-68.
[13] 程平,张洋子,马翾,陈旭,朱保庆,许文涛. 刺激响应型DNA水凝胶的性质及其应用 *[J]. 中国生物工程杂志, 2020, 40(3): 132-143.
[14] 郭胜楠, 李信晓, 王峰, 刘昆梅, 丁娜, 扈启宽, 孙涛. 海马与新皮质组织特异性GABRG2基因敲除小鼠模型的构建及其在遗传性癫痫伴热性惊厥附加症中的初步研究 *[J]. 中国生物工程杂志, 2020, 40(3): 9-20.
[15] 郭晶,侯占铭. Folpcs1基因对尖孢镰刀菌亚麻专化型的无性繁殖和营养生长的调控 *[J]. 中国生物工程杂志, 2020, 40(3): 48-64.