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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2015, Vol. 35 Issue (5): 55-60    DOI: 10.13523/j.cb.20150508
技术与方法     
原位染色检测慢病毒载体转录通读方法的建立
高越1, 檀硕1,2, 任兆瑞1,3, 张敬之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;
3. 生物治疗协同创新中心 四川大学 华西医院 成都 610041
The Establishment of the Methodology used for In Situ Detecting Lentiviral Vector Transcriptional Read-through
GAO Yue1, TAN Shuo1,2, REN Zhao-rui1,3, ZHANG Jing-zhi1,2
1. Shanghai Institute of Medical Genetics, Children's Hospital of Shanghai.Children's Hospital Affiliated to 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;
3. Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China
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摘要：

慢病毒载体作为基因导入系统已被多次应用于基因治疗试验当中,近年来受到了广泛关注。转录通读是慢病毒载体应用过程中存在的潜在不安全因素之一。为了进一步完善慢病毒载体转录通读的检测方法,使检测结果更加直观准确,在已建立的在转录水平检测其转录通读的基础上,进一步模拟病毒载体整合入基因组的状态,于原病毒载体3'-LTR 后插入经密码子优化的LacZ报告基因。经转染细胞后进行原位染色,发生通读现象的细胞会被染成蓝色,从而在蛋白质水平直观地体现慢病毒载体的通读情况,说明所建立的方法是准确可行的。所得结论与qRT-PCR方法在转录水平检测的结果是平行相一致。原位染色检测法的建立为慢病毒载体转录通读的检测、提高慢病毒载体生物安全性的研究提供了技术支持。
关键词： 慢病毒载体;  转录通读;  原位染色;  原病毒载体    
Abstract:

As a gene delivery system, Lentiviral vector (LVV) has been being increasingly used in gene therapy trials, and drawn large attention in recent years. Yet, transcriptional read-through (TRT) is one of potential risks in its application. For the purpose of improving the detection methods of TRT, and making the results intuitive and accurate, a codon optimized reporter gene, LacZ, was placed downstream to the 3'- LTR of the proviral vector to simulate its state in chromosome. Upon the transfection followed with LacZ staining, the cells with TRT phenomenon would be stained blue, and the TRT rate would therefore be visualized at the translational level. Correlating well with the outcomes of qRT-PCR, the established methodology could reflect the feature of TRT, which paves a way for improving LVV biosafety for clinical applications.
Key words: Lentiviral vector    Transcriptional read-throug    In situ staining    Proviral vector
收稿日期: 2015-02-09 出版日期: 2015-05-25
ZTFLH:  Q78  
基金资助:

国家自然科学基金资助项目(81271690)
通讯作者: 张敬之     E-mail: jzhang38@hotmail.com
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引用本文:

高越, 檀硕, 任兆瑞, 张敬之. 原位染色检测慢病毒载体转录通读方法的建立[J]. 中国生物工程杂志, 2015, 35(5): 55-60.

GAO Yue, TAN Shuo, REN Zhao-rui, ZHANG Jing-zhi. The Establishment of the Methodology used for In Situ Detecting Lentiviral Vector Transcriptional Read-through. China Biotechnology, 2015, 35(5): 55-60.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150508    或    https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I5/55


[1] Buchschacher G L Jr, Wong-Staal F. Development of lentiviral vectors for gene therapy for human diseases. Blood, 2000, 95(8): 2499-2504.

[2] Zufferey R, Dull T, Mandel R J, et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol, 1998, 72(12): 9873-9880.

[3] Zaiss A K, Son S, Chang L J. RNA 3' readthrough of oncoretrovirus and lentivirus: implications for vector safety and efficacy. Journal of Virology, 2002, 76(14): 7209-7219.

[4] Higashimoto T, Urbinati F, Perumbeti A, et al. The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors. Gene Ther, 2007, 14(17): 1298-1304.

[5] Schambach A, Galla M, Maetzig T, et al. Improving transcriptional termination of self-inactivating gamma-retroviral and lentiviral vectors. Mol Ther, 2007, 15(6): 1167-1173.

[6] Fang Y, Gong X, Xu M, et al. A self-deletion lentiviral vector to reduce the risk of replication-competent virus formation. J Gene Med, 2013, 15(2): 102-112.

[7] 何佳平, 方彧聃, 张帆, 等. 慢病毒载体转录通读率检测方法的建立. 生物工程学报, 2013, 29(007): 1006-1015. He J P, Fang Y D, Zhang F, et al. Establishment of the methodology for quantifying lentiviral vector transcriptional read-through rate. Chinese Journal of Biotechnology, 2013, 29(007): 1006-1015.

[8] Galy A, Thrasher A J. Gene therapy for the Wiskott-Aldrich syndrome. Curr Opin Allergy Clin Immunol, 2011, 11(6): 545-550.

[9] Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med, 2003, 348(3): 255-256.

[10] Wu X, Li Y, Crise B, et al. Transcription start regions in the human genome are favored targets for MLV integration. Science, 2003, 300(5626): 1749-1751.

[11] Wiley J. Vectors Used in Gene Therapy Clinical Trials.
[2015-02-06]. http://www.wiley.com//legacy/wileychi/genmed/clinical/.

[12] Biffi A, Montini E, Lorioli L, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science, 2013: 341(6148):1233158.

[13] Cavazzana-Calvo M, Payen E, Negre O, et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature, 2010, 467(7313): 318-322.

[14] Aiuti A, Biasco L, Scaramuzza S, et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science, 2013: 341(6148):1233151.

[15] Schambach A, Baum C. Clinical application of lentiviral vectors - concepts and practice. Curr Gene Ther, 2008, 8(6): 474-482.

[16] Escors D and Breckpot K. Lentiviral vectors in gene therapy: their current status and future potential. Arch Immunol Ther Exp(Warsz), 2010, 58(2): 107-119.

[17] Dropulic B. Lentiviral vectors: their molecular design, safety, and use in laboratory and preclinical research. Hum Gene Ther, 2011, 22(6): 649-657.

[18] Pauwels K, Gijsbers R, Toelen J, et al. State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations. Curr Gene Ther, 2009, 9(6): 459-474.

[19] Yang Q, Lucas A, Son S, et al. Overlapping enhancer/promoter and transcriptional termination signals in the lentiviral long terminal repeat. Retrovirology, 2007, 4: 4.
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