CN104962523A - 一种测定非同源末端连接修复活性的方法 - Google Patents

一种测定非同源末端连接修复活性的方法 Download PDF

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CN104962523A
CN104962523A CN201510478296.2A CN201510478296A CN104962523A CN 104962523 A CN104962523 A CN 104962523A CN 201510478296 A CN201510478296 A CN 201510478296A CN 104962523 A CN104962523 A CN 104962523A
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end joining
homologous end
nhej
plasmid
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刘芬菊
杜杰
俞家华
张昊文
尚增甫
张钰烁
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Suzhou University
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Abstract

本发明公开了一种测定非同源末端连接修复活性的方法，该方法采用基因定点突变技术来对HPRT基因进行突变，之后需要质粒转染、药物处理、6-TG处理步骤，最后通过简单的细胞活力检测来观察NHEJ修复的活性，这种方法可以通过检测细胞活力来观察细胞NHEJ修复的活性水平，可用于筛选不同药物、不同基因对NHEJ修复的作用，测定不同细胞系对NHEJ活性抑制药物和基因的反应，从而可以发现具有化疗和放疗增敏效果的药物和基因。

Description

一种测定非同源末端连接修复活性的方法
技术领域
本发明涉及一种测定非同源末端连接修复活性的方法，属于分子生物学领域。
背景技术
非同源末端连接(Non-homologous end joining,NHEJ)是一种细胞修复DNA双链断裂的通路，该修复过程不需要模版染色体的参与，能够直接重新连接两个断裂的DNA末端。然而与另外一种同源重组(Homologous recombination,HR)修复不同，NHEJ修复往往伴随着核苷酸的插入或缺失，从而使基因发生突变，失去基因的功能。
DNA作为遗传信息的保存者，其完整性对细胞来说至关重要。多种内源或外源因素都能造成细胞基因组DNA损伤,细胞内具有应对不同形式损伤的复杂的修复系统。其中DNA双链断裂(DNA double-strand breaks,DSBs)作为最严重最致命的损伤形式，如果没有被及时修复，将导致细胞死亡。在肿瘤的临床治疗中，放疗和多数化疗药物可以导致肿瘤细胞DNA双链断裂，肿瘤细胞主要通过NHEJ和HR修复来对断裂的DNA双链进行修复，其中NHEJ不存在细胞周期依赖性，是细胞中修复DNA双链断裂的主要方式，其活性在一定程度上决定了肿瘤细胞对电离辐射和化疗药物的敏感性，当肿瘤细胞中的NHEJ修复活性被抑制后，放疗和化疗的效果能得到明显的改善。
目前可以用来测定NHEJ活性的方法主要有DNA双链断裂特异性蛋白γH2AX和53BP1染色，DNA断裂片段的中性彗星电泳，以及采用比较复杂的分子生物学手段来研究NHEJ的活性，如在大范围核酸酶I-SceI基础上设计的针对NHEJ修复活性的报告质粒，转染细胞后筛选稳定表达细胞株，再引入I-SceI核酸酶进行DNA双链断裂的诱导，最后通过观察报告质粒的荧光表达来反应NHEJ修复的能力(Identification of Novel Radiosensitizers in a High-Throughput,Cell-Based Screen for DSB Repair Inhibitors，Alexander G，《Molecular CancerTherapeutics》第14卷，第326-342页，2015年2月)。但是上述这些方法比较复杂且步骤繁多，而且需要一些特异性的染色以及对特定细胞株的筛选。
基因组定点编辑技术目前发展迅速，现在主要有三种类型，锌指核酸内切酶(Zinc finger nucleases,ZFNs)，类转录激活因子效应物核酸酶(Transcriptionactivator-like effector nucleases，TALENs)，规律性重复短序列丛集及其系统(Clustered regularly interspaced short palindromic repeats,CRISPR/CRISPRassociated system,Cas9)，这些技术可以对基因组中特定的DNA位点进行切断，通过诱导NHEJ或HR修复来达到对基因定点编辑的目的，而且突变效率高、制作简单、成本较低。因此如何利用基因组定点编辑技术来简明的测定非同源末端连接修复活性成为当前亟待解决的问题。
发明内容
为解决上述技术问题，本发明的目的是提供一种测定非同源末端连接修复活性的方法。
次黄嘌呤鸟嘌呤磷酸核糖转移酶(Hypoxanthine-guanine phosphoriboSyltransferase,HPRT)参与细胞内嘌呤核苷酸生物合成的补救途径，6-硫代鸟嘌呤(6-Thioguanine，6-TG)能在该酶的作用下掺入DNA中，从而抑制DNA合成，使细胞无法存活。如果用上述基因定点编辑技术在HPRT基因上造成DNA双链断裂，当HPRT基因通过NHEJ修复发生突变后，可使细胞在含有6-TG的培养基中存活，而没有发生NHEJ修复而突变的细胞则不能在含有6-TG的培养基中存活，这样存活细胞的数量及活力就反映了NHEJ修复水平的高低。
本发明的原理是采用基因定点突变技术(ZFN，TALEN或CRISPR/Cas9)在HPRT基因上诱导DNA双链断裂，当HPRT基因的断裂点发生NHEJ修复时，会发生基因突变，导致细胞内HPRT功能失活。正常细胞在HRPT失活后并不会影响细胞的功能，但是在含有6-TG的培养基中，6-TG可以在HPRT的作用下掺入DNA引起细胞死亡，而发生NHEJ后突变的细胞由于HPRT基因功能失活，可以抵抗6-TG的毒性而存活下来，最终可以通过检测细胞的活力来了解细胞的NHEJ修复活性。
本发明的技术方案是：
一种测定非同源末端连接修复活性的方法，包括下述步骤：
一种测定非同源末端连接修复活性的方法，包括下述步骤：
(1)采用TALEN技术或CRISPR/Cas9技术构建针对次黄嘌呤鸟嘌呤磷酸核糖转移酶基因的质粒；
(2)对(1)中构建所得的质粒结合哺乳动物细胞采用非同源末端连接修复分子抑制剂进行处理，得到经抑制处理的哺乳动物细胞；
(3)将(2)中所得的哺乳动物细胞用含有6-硫代鸟嘌呤的DMEM培养基进行培养；
(4)在经(3)处理好的哺乳动物细胞中加入MTT溶液，待蓝色甲臜颗粒形成后用DMSO溶解，然后采用酶标仪测定其在OD570nm的吸光值。
其进一步的技术方案是：
步骤(1)中采用TALEN技术构建针对次黄嘌呤鸟嘌呤磷酸核糖转移酶基因的质粒时，所设计打靶基因的左臂识别序列为SEQ ID NO:1，设计打靶基因的右臂识别序列为SEQ ID NO:2。
步骤(1)中采用CRISPR/Cas9技术构建针对次黄嘌呤鸟嘌呤磷酸核糖转移酶基因的质粒时，所设计的靶点引物的正向引物序列为SEQ ID NO:3，所设计的靶点引物的反向引物序列为SEQ ID NO:4。
步骤(2)中所述哺乳动物细胞为293T细胞，所述非同源末端连接修复分子抑制剂为非同源末端连接修复分子DNA-PK的抑制剂NU7441和DNA-PKcssiRNA中的一种，且所述DNA-PKcs siRNA的基因序列为SEQ ID NO:5。
步骤(2)中对(1)中构建所得的质粒结合哺乳动物细胞采用非同源末端连接修复分子抑制剂进行处理是指：将步骤(1)中构建所得的质粒转染293T细胞并对转染后的293T细胞采用非同源末端连接修复分子DNA-PK抑制剂NU7441处理。
步骤(2)中进行质粒转染的过程包括：将293T细胞接种在培养皿中，当该细胞密度达到70％时，采用Lipofectamine 3000作为转染试剂将(1)中构建所得的质粒转染293T细胞。
步骤(2)中对(1)中构建所得的质粒结合哺乳动物细胞采用非同源末端连接修复分子抑制剂进行处理是指：将步骤(1)中构建所得的质粒和DNA-PKcssiRNA共转染293T细胞。
步骤(2)中进行质粒转染的过程包括：将293T细胞接种在培养皿中，当该细胞密度达到70％时，采用Lipofectamine 3000作为转染试剂将(1)中构建所得的质粒和DNA-PKcs siRNA共转染293T细胞。
借由上述方案，本发明至少具有以下优点：本发明所述方法采用基因定点突变技术来对HPRT基因进行突变，之后需要质粒转染、药物处理、6-TG处理步骤，最后通过简单的细胞活力检测来观察NHEJ修复的活性，这种方法可以通过检测细胞活力来观察细胞NHEJ修复的活性水平，可用于筛选不同药物、不同基因对NHEJ修复的作用，测定不同细胞系对NHEJ活性抑制药物和基因的反应，从而可以发现具有化疗和放疗增敏效果的药物和基因。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是本发明测定NHEJ活性的方法的技术方案图；
图2为本发明中不同浓度NU7441对NHEJ修复活性的影响；
图3为本发明中2.0μmol/L NU7441处理不同时间对NHEJ修复活性的影响；
图4为本发明中DNA-PKcs siRNA转染对NHEJ修复活性的影响。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述，以下实施例用于说明本发明，但不用来限制本发明的范围。
下述具体实施例中TALEN构建试剂盒购自斯丹赛生物技术有限公司，CRISPR/Cas9构建试剂盒购自唯尚立德生物技术有限公司，NHEJ修复分子DNA-PK抑制剂NU7441购自Selleck公司，DNA-PKcs siRNA由吉玛基因公司合成，引物由invitrogen上海公司合成，用蒸馏水配置成10μmol/L，转染试剂Lipofectamine 3000购自invitrogen上海公司，MTT购自上海生工生物工程公司。
具体实施例一：
采用TALEN技术测定不同浓度NU7441对NHEJ的抑制作用，其具体步骤为：
(1)构建针对HPRT基因的TALEN质粒。具体步骤为：按照TALEN设计原则设计打靶HPRT基因的左臂和右臂识别序列，左臂识别序列：ATGACCTTGATTTA(SEQ ID NO:1)，右臂识别序列：CCAAATCCTCAGCA(SEQ ID NO:1)，按照TALEN构建试剂盒的方法，将两个识别序列插入相应的骨架载体，转化感受态大肠杆菌，均匀涂布于卡那霉素抗性(20μg/ml)的平板中，置于37℃培养箱中培养12～16h后挑选3～5个单克隆接种于5ml LB培养液(含卡那霉素20μg/ml)，置于37℃250rpm的摇床中培养16h，抽提质粒测序，并与设计的识别序列比对，获取测序正确的左臂和右臂TALEN质粒。
(2)将左臂和右臂TALEN质粒转染293T细胞。具体步骤为：将293T细胞接种在6cm培养皿中，当细胞密度达到70％时，采用Lipofectamine 3000将4μg左臂质粒和4μg右臂质粒转染293T细胞。
(3)不同浓度的抑制剂NU7441处理293T细胞。具体步骤为：将TALEN质粒转染24h后的细胞接种至96孔板，待细胞贴壁后分别用0，0.1，0.5，1.0，2.0μmol/L的NU7441处理4h。
(4)6-TG处理细胞。具体步骤为：在NU7441处理结束后，用含有30μmol/L6-TG的DMEM培养基培养293T细胞72h。
(5)MTT法测定细胞活力。具体步骤为：处理结束后加入MTT溶液，2h后蓝色甲臜颗粒形成，用DMSO溶解后用酶标仪测定各孔在OD570nm处的吸光值。细胞活力反映了NHEJ修复的活力，空白对照组设为NHEJ活力为100％。
实验结果如图2所示，在293T细胞中，NU7441浓度在0.5，1.0μmol/L时处理4h，对NHEJ修复活性的抑制效果最佳，抑制率在50％左右，当浓度提高到2.0μmol/L时，抑制效果反而减弱。
具体实施例二：
采用CRISPR/Cas9技术测定2.0μmol/L NU7441对NHEJ抑制作用的时间依赖性，其具体步骤为：
(1)构建针对HPRT基因的Cas9/gRNA质粒。具体步骤为：按照gRNA设计原则设计HPRT靶点引物，正向引物：AAACACCGAAAGGGTGTTTATTCCTCA(SEQ ID NO:3)，反向引物：CTCTAAAACTGAGGAATAAACACCCTTT(SEQ ID NO:4)，将引物退火形成二聚体，按照CRISPR/Cas9构建试剂盒的方法，将gRNA序列引物二聚体插入Cas9/gRNA质粒，转化感受态大肠杆菌，涂于氨苄抗性的平板，挑选3～5个单克隆摇菌，提取质粒进行测序并与设计的识别序列比对，获得测序正确的Cas9/gRNA质粒。
(2)将Cas9/gRNA质粒转染293T细胞。具体步骤为：将293T细胞接种在6cm培养皿中，当细胞密度达到70％时，采用Lipofectamine 3000将5μgCas9/gRNA质粒转染293T细胞。
(3)2.0μmol/L NU7441处理293T细胞。具体步骤为：将Cas9/gRNA质粒转染24h后的细胞接种至96孔板，待细胞贴壁后分别用2.0μmol/L的NU7441分别处理0，0.5，1，2，4h。
(4)6-TG处理细胞。具体步骤为：在NU7441处理结束后，用含有30μmol/L6-TG的DMEM培养基培养293T细胞72h。
(5)MTT法测定细胞活力。具体步骤为：处理结束后加入MTT溶液，2h后蓝色甲臜颗粒形成，用DMSO溶解后用酶标仪测定各孔在OD570nm处的吸光值。细胞活力反应了NHEJ修复的活力，空白对照组设为NHEJ活力为100％。
实验结果如图3所示，在293T细胞中，2.0μmol/L NU7441处理1h对NHEJ修复活性的抑制效果最佳，抑制率在50％左右，随着处理时间的进一步延长，对NHEJ活性的抑制作用减弱。
具体实施例三：
采用CRISPR/Cas9技术测定DNA-PKcs siRNA对NHEJ的抑制作用，其具体步骤为：
(1)构建针对HPRT基因的Cas9/gRNA质粒，其具体步骤参见具体实施例二中(1)所述。
(2)将Cas9/gRNA质粒和DNA-PKcs siRNA共转染293T细胞。其具体步骤为：将293T细胞接种在6cm培养皿中，当细胞密度达到70％时，采用Lipofectamine 3000将3μg Cas9/gRNA质粒和3μg DNA-PKcs siRNA共转染293T细胞，对照组转染阴性对照siRNA。其中DNA-PKcs siRNA的基因序列为UUCUCCGAACGUGUCACGUTT(SEQ ID NO:5)。
(3)6-TG处理细胞。其具体步骤为：转染24h后细胞接种至96孔板，当细胞贴壁后将培养基换成含有30μmol/L 6-TG的DMEM培养基，继续培养72h。
(4)MTT法测定细胞活力。其具体步骤为：处理结束后加入MTT溶液，2h后蓝色甲臜颗粒形成，用DMSO溶解后用酶标仪测定各孔在OD570nm的吸光值。细胞活力反应了NHEJ修复的活力，对照组设为NHEJ活力为100％。
实验结果如图4所示，用siRNA转染降低NHEJ修复关键分子DNA-PKcs的表达后，293T细胞中NHEJ修复活性受到了明显的抑制。
综上，本发明所述方法可以通过检测细胞活力来观察细胞NHEJ修复的活性水平。这种方法需要基因定点突变技术来对HPRT基因进行突变，之后需要质粒转染、药物处理、6-TG处理步骤，最后通过简单的细胞活力检测来观察NHEJ修复的活性，这种方法可以用来筛选不同药物、不同基因对NHEJ修复的作用，可以测定不同细胞系对NHEJ活性抑制药物和基因的反应，从而可以发现具有化疗和放疗增敏效果的药物和基因。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。

Claims (8)

1.一种测定非同源末端连接修复活性的方法，其特征在于：包括下述步骤：
(1)采用TALEN技术或CRISPR/Cas9技术构建针对次黄嘌呤鸟嘌呤磷酸核糖转移酶基因的质粒；
(2)对(1)中构建所得的质粒结合哺乳动物细胞采用非同源末端连接修复分子抑制剂进行处理，得到经抑制处理的哺乳动物细胞；
(3)将(2)中所得的哺乳动物细胞用含有6-硫代鸟嘌呤的DMEM培养基进行培养；
(4)在经(3)处理好的哺乳动物细胞中加入MTT溶液，待蓝色甲臜颗粒形成后用DMSO溶解，然后采用酶标仪测定其在OD570nm的吸光值。
2.根据权利要求1所述的测定非同源末端连接修复活性的方法，其特征在于：步骤(1)中采用TALEN技术构建针对次黄嘌呤鸟嘌呤磷酸核糖转移酶基因的质粒时，所设计打靶基因的左臂识别序列为SEQ ID NO:1，设计打靶基因的右臂识别序列为SEQ ID NO:2。
3.根据权利要求1所述的测定非同源末端连接修复活性的方法，其特征在于：步骤(1)中采用CRISPR/Cas9技术构建针对次黄嘌呤鸟嘌呤磷酸核糖转移酶基因的质粒时，所设计的靶点引物的正向引物序列为SEQ ID NO:3，所设计的靶点引物的反向引物序列为SEQ ID NO:4。
4.根据权利要求1所述的测定非同源末端连接修复活性的方法，其特征在于：步骤(2)中所述哺乳动物细胞为293T细胞，所述非同源末端连接修复分子抑制剂为非同源末端连接修复分子DNA-PK的抑制剂NU7441和DNA-PKcssiRNA中的一种，且所述DNA-PKcs siRNA的基因序列为SEQ ID NO:5。
5.根据权利要求4所述的测定非同源末端连接修复活性的方法，其特征在于：步骤(2)中对(1)中构建所得的质粒结合哺乳动物细胞采用非同源末端连接修复分子抑制剂进行处理是指：将步骤(1)中构建所得的质粒转染293T细胞并对转染后的293T细胞采用非同源末端连接修复分子DNA-PK抑制剂NU7441处理。
6.根据权利要求5所述的测定非同源末端连接修复活性的方法，其特征在于：步骤(2)中进行质粒转染的过程包括：将293T细胞接种在培养皿中，当该细胞密度达到70％时，采用Lipofectamine 3000作为转染试剂将(1)中构建所得的质粒转染293T细胞。
7.根据权利要求4所述的测定非同源末端连接修复活性的方法，其特征在于：步骤(2)中对(1)中构建所得的质粒结合哺乳动物细胞采用非同源末端连接修复分子抑制剂进行处理是指：将步骤(1)中构建所得的质粒和DNA-PKcssiRNA共转染293T细胞。
8.根据权利要求7所述的测定非同源末端连接修复活性的方法，其特征在于：步骤(2)中进行质粒转染的过程包括：将293T细胞接种在培养皿中，当该细胞密度达到70％时，采用Lipofectamine 3000作为转染试剂将(1)中构建所得的质粒和DNA-PKcs siRNA共转染293T细胞。
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