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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2017, Vol. 37 Issue (2): 74-80    DOI: 10.13523/j.cb.20170211
技术与方法     
单细胞PCR体系的建立及其在CRISPR/Cas9靶点活性检测中的应用
徐振宇1,2, 任红艳2, 毕延震2, 郑新民2, 李莉2, 张佳兰1
1. 长江大学动物科学学院 荆州 434025;
2. 湖北省农业科学院畜牧兽医研究所 动物胚胎工程与分子育种湖北省重点实验室 武汉 430064
Establishment of the Single-cell PCR System and Its Application in the Target-activity Detection of CRISPR/Cas9 System
XU Zhen-yu1,2, REN Hong-yan2, BI Yan-zhen2, ZHENG Xin-min2, LI Li2, ZHANG Jia-lan1
1. College of Animal Sciences, Yangtze University, Jingzhou 434025, China;
2. Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province, Institute of Animal and Veterinary Sciences, Hubei Academy of Agricultural Sciences, Wuhan 430064, China
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摘要:

单细胞PCR是以单个细胞所含的DNA或RNA为模板进行扩增,因此其模板的制备是影响整个反应成功与否的关键因素。利用三种不同蛋白酶K细胞裂解液(SDS细胞裂解液、NP-40细胞裂解液、Tween-20细胞裂解液)分别对体外培养成熟的单个猪卵母细胞进行裂解,并直接作为模板进行PCR扩增,结果表明:NP-40细胞裂解液制备的模板质量最好,其扩增效率为95%;其次为Tween-20细胞裂解液,其扩增效率为65%;SDS细胞裂解液产物中未检测到阳性条带。结合显微注射技术对sgRNA靶点活性进行检测,结果表明利用单细胞PCR结合显微注射的方法,在受精卵内对CRISPR/Cas9靶点活性进行检测确实可行,检测结果真实可靠。

关键词: 基因修饰动物细胞裂解液靶点活性检测单细胞PCR    
Abstract:

Single-cell PCR uses the DNA or RNA contained in a single cell as template for the amplification.The quality of the template is one of the key factors for the success of the reaction.Three different proteinase K cell lysate buffers (SDS cell lysate buffer,NP-40 cell lysate buffer and Tween-20 cell lysate buffer) were used to digest the matured oocytes of porcine in vitro.The product of digestion was used as the template for single-cell PCR amplification directly.The results showed that the quality of template processed by NP-40 cell lysates buffer was the best,with a high amplification efficiency of 95%.The amplification efficiency of PCR with template produced by Tween-20 cell lysates buffer was about 65%;while there was no positive bands were detected using the SDS cell lysates Buffer.Combined the single-cell PCR with the microinjection techniques,and it has been used to detect the activity of CRISPR-Cas9 target in vitro.The results showed that this detection method has a high efficiency and stability.

Key words: Cell lysates Buffer    Activity detection of target    Genetic modified animal    Single-cell PCR
收稿日期: 2016-08-03 出版日期: 2017-02-25
ZTFLH:  Q819  
基金资助:

转基因生物新品种培育重大专项(2016ZX08006001-005,2016ZX08006002-006,2016ZX08010003-006)、湖北省农业科技创新中心(2016-620-000-001-027)、湖北省科技支撑计划(2014BBB010)资助项目

通讯作者: 任红艳, 张佳兰     E-mail: renhongyan507@163.com;zjlgpy88@163.com
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引用本文:

徐振宇, 任红艳, 毕延震, 郑新民, 李莉, 张佳兰. 单细胞PCR体系的建立及其在CRISPR/Cas9靶点活性检测中的应用[J]. 中国生物工程杂志, 2017, 37(2): 74-80.

XU Zhen-yu, REN Hong-yan, BI Yan-zhen, ZHENG Xin-min, LI Li, ZHANG Jia-lan. Establishment of the Single-cell PCR System and Its Application in the Target-activity Detection of CRISPR/Cas9 System. China Biotechnology, 2017, 37(2): 74-80.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170211        https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I2/74

[1] Jeffreys A J,Wilson V,Neumann R,et al.Amplification of human minisatellites by the polymerase chain reaction:towards DNA fingerprinting of single cells.Nucleic Acids Research,1988,16(23):10953-10971.
[2] Kumar R,Barbacid M.Oncogene detection at the single cell level.Oncogene,1988,3(3):647-651.
[3] 刘建强,吴大洲,张印则,等.单细胞巢式PCR检测RHD的初步研究.中国输血杂志,2015,28(7):760-762. Liu J Q,Wu D Z,Zhang Y Z,et al.Primary study on single-cell nested PCR for detection of RHD.Chinese Journal of Blood Transfusion,2015,28(7):760-762.
[4] 钟昌高,李麓芸,陆长富,等.单细胞单轮二重PCR诊断X-连锁鱼鳞病.生命科学研究,2009,13(4):332-336. Zhong C G,Li L Y,Lu C F,et al.Diagnosing X-linked ichthyosis by monoplast single-round duplex PCR.Life Science Research,2009,13(4):332-336.
[5] Chrenek P,Boulanger L,Heyman Y,et al.Sexing and multiple genotype analysis from a single cell of bovine embryo.Theriogenology,2001,55(5):1071-1081.
[6] 陈从英,黄路生,陈静波,等.牛早期胚胎性别鉴定PCR反应体系的优化研究.畜牧兽医学报,2003,34(3):209-212. Chen C Y,Huang L S,Chen J B,et al.The study on optimizing the system of PCR for sex determination of bovine preimplantation embryos.Acta Veterinaria et Zootechnica Sinica,2003,34(3):209-212.
[7] Piyamongkol W,Harper J C,Sherlock J K,et al.A successful strategy for preimplantation genetic diagnosis of myotonic dystrophy using multiplex fluorescent PCR.Prenat Diagn,2001,21:223-232.
[8] Abou-Sleiman P M,Apessos A,Harper J C,et al.First application of preimplantation genetic diagnosis to neurofibromatosis type 2 (NF2).Prenat Diagn,2002,22:519-524.
[9] Sandalinas M,Sadowy S,Alikani M,et al.Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage.Human Reprod,2001,16:1954-1958.
[10] Bahe M,Escudero T,Sandalinas M,et al.Improvements of preimplantation diagnosis of aneuploidy by using microwave-hybridization,cell recycling and monocolor labeling of probes.Molec Human Reprod,2000,9:849-854.
[11] Copeland N G,Jenkins N A.Harnessing transposons for cancer gene discovery.Nature Reviews Cancer,2010,10:696-706.
[12] Kool J,Berns A.High-throughput insertional mutagenesis screens in mice to identify oncogenic networks.Nature Reviews Cancer,2009,1038(10):389-399.
[13] Wang H Y,Yang H,Shivalila C,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-Mediated genome engineering.Cell,2013,153(4):910-918.
[14] Bogdanove A J,Voytas D F.TAL effectors:customizable proteins for DNA targeting.Science,2011,333(6051):1843-1846.
[15] Carroll D,Beumer K J,Morton J J,et al.Gene targeting in Drosophila and Caenorhabditis elegans with zinc-finger nucleases.Humana Press,2008,435:63-77.
[16] Urnov F D,Rebar E J,Holmes M C,et al.Genome editing with engineered zinc finger nucleases.Nature Reviews Genet,2010,11(9):636-646.
[17] Sorek R,Kunin V,Hugenholtz P.CRISPR-A widespread system that provides acquired resistance against phages in bacteria and archaea.Nature Reviews Microbiology,2008,6(3):181-186.
[18] Wiedenheft B,Stenberg S H,Doudna J A. RNA-guided genetic silencing systems in bacteria and archaea.Nature,2012,482(7385):331-338.
[19] 左其生,李 东,张亚妮,等.CRISPR-CAS介导的基因编辑工具.生物技术通报,2014,7:37-43. Zuo Q S,Li D,Zhang Y N,et al.Gene editing tools mediated by CRISPR-Cas.Biotechnology Bulletin,2014,7:37-43.
[20] Sermon K,Lissens W,NagyZP,et al.Simultaneous amplification of the two most frequent mutations of infantile Tay-Sachs disease in single blastomeres.Human Reproduction,1995,10(8):2214-2217.
[21] GitlinSA,LanzendorfSE,GibbonsWE.Polymerase chain reaction amplification specificity:incidence of allele dropout using different DNA preparation methods for heterozygous single cells.Assist Reprod Genet,1996,13(2):107-111.
[22] Dietmair W,Hartmann A,Wallinger S,et al.Mutliple mutation anlyses in single tumor cells with improved whole genome amplification.American Journal of Pathology,1999,154(1):83-95.
[23] 易萍,李力,王华,等.细胞裂解法对单细胞PCR 扩增效率的影响.中国优生与遗传杂志,2005,13(2):27-32. Yi P,Li L,Wang H,et al.The assessment of the amplified efficacy of single cell PCR by different lysis buffers.Chinese Journal of Birth Health & Heredity,2005,13(2):27-32.
[24] Sorek R,Kunin V,Hugenholtz P.CRISPR-A widespread system that provides acquired resistance against phages in bacteria and archaea.Microbiology,2008,3(6):181-186.
[25] 李晓敏,任红艳,毕延震,等.CRISPR/Cas9体外酶切检测猪生长抑素基因定点修饰靶点活性的研究,中国畜牧兽医,2016,43(1):31-38. Li X M,Ren H Y,Bi Y Z,et al.Study on detection of pig SST gene site-directed modification activity by CRISPR/Cas9 system in vitro.China Animal Husbandry and Veterinary Medicine,2016,43(1):31-38.

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