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CN108504657A - 利用crispr-cas9技术敲除hek293t细胞kdm2a基因的方法 - Google Patents

利用crispr-cas9技术敲除hek293t细胞kdm2a基因的方法 Download PDF

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CN108504657A
CN108504657A CN201810325470.3A CN201810325470A CN108504657A CN 108504657 A CN108504657 A CN 108504657A CN 201810325470 A CN201810325470 A CN 201810325470A CN 108504657 A CN108504657 A CN 108504657A
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彭勇波
许文豪
梁大焱
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South Central Minzu University
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Abstract

本发明提供了一种CRISPR‑CAS9技术敲除HEK293T细胞KDM2A基因的方法，步骤如下：(1)、细胞转染；(2)、细胞基因组提取；(3)、PCR鉴定；(4)、基因水平验证；(5)、蛋白水平验证：在基因水平验证突变细胞株后，用蛋白质免疫印迹(Western blotting)进一步验证K3、K7‑1和K7‑2是KDM2A基因缺陷型。本发明还同时提供了利用上述方法制备的KDM2A基因敲除的HEK293T细胞系及以其作为细胞模型在研究KDM2A甲基化酶所涉及的细胞增殖和凋亡信号通路中的应用。

Description

利用CRISPR-CAS9技术敲除HEK293T细胞KDM2A基因的方法
技术领域
本发明提供了一种利用CRISPR-CAS9技术敲除HEK293T细胞KDM2A基因的方法，属于基因工程技术领域。
背景技术
HEK细胞是来源于人源胚胎肾细胞，HEK293T细胞是HEK细胞的变种，是由其转染SV40大T抗原得到的可以体外增殖的细胞系，这种细胞系增殖快，转染效率高，常用于蛋白表达。赖氨酸依赖性去甲基化酶家族，对细胞增殖和凋亡具有极为重要的影响，这一特征决定了它可以作为肿瘤治疗的潜在应用。细胞癌变的显著特征之一就是甲基化酶和去甲基化酶的表达失调而引发的表观遗传调控功能紊乱。在该去甲基化酶家族中，KDM2A是截至目前研究仍不甚清楚的一个成员。KDM2A的主要功能是识别组蛋白H3的36号赖氨酸(H3K36)甲基化位点，使其去甲基化，使异染色质结构稳定并促进该区域的基因表达，以发挥该基因的原癌基因功能。现有研究表明，多种癌症的发生均与其表达上调有关，如肠癌、肺癌和乳腺癌等。现有研究表明，KDM2A能够促进细胞生长和迁移，但是对于它如何影响细胞凋亡仍不清楚。因此，构建KDM2A的敲除细胞系，并以此用于细胞凋亡信号通路的研究是极为重要的，构建成功的细胞系可用于蛋白水平表达、转录组分析以及其他多种实验用途。虽然目前有部分关于KDM2A基因沉默以及RNA干扰的相关报道，但是与基因敲除相比，基因敲除的效果更为彻底，更有利于研究KDM2A对细胞增殖和凋亡作用的影响。
发明内容
本发明解决了现有技术中的不足，提供了一种CRISPR-CAS9技术敲除HEK293T细胞KDM2A基因的方法。本发明还同时提供了利用上述方法制备的KDM2A基因敲除的HEK293T细胞系及以其作为细胞模型在研究KDM2A甲基化酶所涉及的细胞增殖和凋亡信号通路中的应用。
实现本发明上述目的所采用的技术方案为：
一种基于CRISPR/CAS9系统的特异性敲除人源KDM2A基因的sgRNA，所述sgRNA在KDM2A基因上的靶序列位于KDM2A基因(NM_012308.2)的14号和18号外显子上，其中14号外显子上的靶序列为：5’-ACATCGCACTCGTCTCCGTC-3’，18号外显子上的靶序列为：5’-TTTGAAGCGCTCATCGCGGC-3’。
本发明同时提供了上述sgRNA的表达载体pX458-KDM2A。所述表达载体pX458-KDM2A的构建方法如下，在14号外显子上以及18号外显子上的靶序列的5’端加上BbsI的酶切位点caccg，依次构成序列SEQ ID NO：1和SEQ ID NO：2，并人工合成其互补序列SEQ IDNO：3和SEQ ID NO：4，将互补序列SEQ ID NO.1和SEQ ID NO：3，以及SEQ ID NO.2和SEQ IDNO：4所形成的双链DNA，与经过BbsI酶切的pX458载体连接，从而制得表达载体pX458-KDM2A。
进一步的，本发明提供了一种KDM2A基因敲除的HEK293T细胞系以及所述的KDM2A基因敲除的HEK293T细胞系的构建方法，包括以下步骤：以表达载体pX458-KDM2A作为KDM2A基因的打靶载体，转染HEK293T细胞，获得的KDM2A基因敲除的单克隆细胞株，分别简记为K3，K7-1和K7-2。
具体的构建方法如下：
(1)、细胞转染：培养HEK293T细胞至70～90％汇合度时准备转染，将含有表达载体pX458-KDM2A的混合液加入HEK293T细胞中进行转染，然后培养；
(2)、细胞基因组提取：将转染后的HEK293T细胞进行基因组提取；
(3)、PCR鉴定：根据KDM2A基因序列，设计引物KDM2A-3F和KDM2A-3R、KDM2A-7F和KDM2A-7R，上述引物的序列分别如：SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示；利用上述引物对所提取的基因组进行PCR反应；
(4)、基因水平验证：将PCR产物测序，测序结果与野生型PCR序列比对，确定K3、K7-1和K7-2为突变体；
(5)、蛋白水平验证：在基因水平验证突变细胞株后，用蛋白质免疫印迹(Westernblotting)进一步验证K3、K7-1和K7-2是KDM2A基因缺陷型。
与现有技术相比，本发明具有以下优点：(1)本发明利用CRISPR/Cas9系统从HEK293T细胞中敲除KDM2A基因，操作简便，效果完整而彻底，与HEK293T细胞特性一起构成了理想的细胞模型，用于研究细胞内表观遗传学的改变。(2)本发明中基于CRISPR/Cas9系统敲除KDM2A基因的方法，与沉默、敲低、干扰等方法相比，敲除效果更加彻底，更有利于研究KDM2A对细胞增殖和凋亡作用的影响。(3)本发明从基因和蛋白水平的检测都证实，KDM2A已被成功敲除，说明该蛋白已被彻底改变，可能造成KDM2A功能的彻底丧失，适于对KDM2A进行更加深入的研究。
附图说明
图1为实施例中sgRNA检测及被转染CRISPR打靶载体的突变型细胞基因型鉴定图；
图2为敲除型细胞株和野生型细胞株的KDM2A蛋白表达含量差异图；
图3、图4及图5为a)、b)、c)三组流式细胞分析野生型和各敲除系之间的细胞增殖和凋亡差异图。
具体实施方式
下面结合具体实施例对本发明做详细具体的说明，以下实施例仅用于说明本发明，但不用于限制本发明的范围。若未特殊指明，实施例均按照常规实验条件进行，如J.萨姆布鲁克等人所著的《分子克隆实验指南》，或按照试剂厂商说明书建议的条件。
实施例1：利用CRISPR-CAS9技术敲除HEK293T细胞KDM2A基因的方法。
1、实验材料
真核表达载体pSpCas9(BB)-2A-GFP(pX458)、人源HEK293T细胞。
2、CRISPR/Cas9打靶载体的构建
(1)靶序列的确定
根据网站http://asia.ensembl.org/index.html获取人源KDM2A基因(NM_012308.2)的外显子信息，选择第14号和第18号外显子作为靶序列。之后设计sgRNA序列，在其5’端加上BbsI酶切位点序列(如SEQ ID NO：1、2所示)，并合成与其互补的序列(如SEQ IDNO：3、4所示)。
(2)靶序列与pX458质粒连接
①pX458空载体酶切，20μL反应体系见表1，37℃酶切过夜。
表1 pX458空质粒酶切体系
成分 用量
pX458 1μg(x μL)
BbsI酶 1μL
NEBuffer 2.1(10×) 2μL
ddH2O 17-x μL
②pX458酶切产物的回收纯化
根据Axygen胶回收试剂盒说明书进行。
③合成的sgRNA序列和互补序列形成双链DNA，10μL反应体系见表2，反应程序：37℃，30min；95℃5min，自然降至室温。
表2合成片段形成双链体系
成分 用量
sgRNA序列(100μM) 1μL
互补序列(100μM) 1μL
T4 Polynucleotide Kinase反应缓冲液 1μL
T4 PNK(NEB) 0.5μL
ddH2O 6.5μL
④将形成双链的目的片段稀释250倍后与载体pX458连接，得到重组载体pX458-KDM2A。10μL反应体系见表3，反应程序：室温连接3h。
表3连接体系
成分 用量
靶序列 1μL(500ng)
胶回收pX458质粒 5μL
10×T4 DNA连接酶缓冲液 1μL
T4 Ligase 1μL
ddH2O 2μL
(3)转化大肠杆菌DH5α，挑取单菌落PCR鉴定
将连接产物5μL加入至50μL DH5α感受态细胞中，混匀后冰浴30分钟，然后在42℃热激转化90秒，随后冰浴2分钟，加入37℃温育好的无菌LB培养基(1％胰蛋白胨、0.5％酵母提取物、1％氯化钠)950μL，在37℃及转速为180转/分的条件下震荡培养2小时，取100μL菌液涂布在氨苄青霉素抗性固体LB培养基(含100μg/mL的氨苄青霉素)上，37℃过夜倒置培养后挑取单菌落，在1mL含100μg/mL氨苄青霉素的液体LB培养基中培养，进行菌液PCR鉴定，筛选出具有正确电泳条带的质粒进行下一步实验。测序引物是U6启动子的正向引物序列：
5’-GAGGGCCTATTTCCCATGATTCC-3’(SEQ ID NO：9)。
3、质粒的制备与鉴定
将上一步筛选出符合预期的克隆，用康为世纪高纯度质粒大提试剂盒大量制备无内毒素的质粒pX458-KDM2A，测定其纯度和浓度。操作步骤按说明书进行。
4、KDM2A基因敲除细胞系的构建
(1)接种HEK293T细胞至70～90％汇合度时准备转染，转染前，细胞转染步骤如下：
①用Opti-MEM培养基稀释Lipofectamine 3000试剂，并充分混匀；
②用Opti-MEM培养基稀释大提好的质粒pX458-KDM2A，制备DNA预混液，然后添加P3000试剂，并充分混匀；
③将以上两种混合液按照1∶1的比例混匀，然后在室温下孵育5分钟，形成DNA-脂质体复合物，并加入到细胞中，在37℃下恒温培养箱中培养4小时后换液；
④培养24小时后，使用分选型流式细胞仪筛选出带有EGFP标记的单克隆细胞在96孔板中培养；
⑤待细胞数量足够多时分别提取基因组DNA进行鉴定。
(2)细胞基因组提取：将贴壁细胞处理为细胞悬液，以2000rpm离心5分钟，弃尽上清，使用康为世纪通用型柱式基因组提取试剂盒，按照试剂盒说明书操作步骤将细胞基因组提取出来。
(3)PCR鉴定：在KDM2A基因上，设计引物KDM2A-3F和KDM2A-3R、KDM2A-7F和KDM2A-7R序列如下。PCR反应条件：95℃预变性5分钟；35个循环：95℃变性30秒，58℃退火30秒，72℃延伸1分钟；最后72℃终延伸7分钟。PCR体系见表5。
KDM2A-3F：5’-TGTAATCTTCTACCCTATCCA-3’(SEQ ID NO：5)
KDM2A-3R：5’-AAGAGGAGGACTCAACCA-3’(SEQ ID NO：6)
KDM2A-7F：5’-TGTCGGTTCAGAGGGTTTG-3’(SEQ ID NO：7)
KDM2A-7R：5’-CCACAGGCTTTCAGATGC-3’(SEQ ID NO：8)
表5PCR扩增体系
成分 用量(μL)
ddH2O 10.5
KDM2A-F 0.5
KDM2A-R 0.5
基因组DNA 1
2×Pfu Mix 12.5
将其中扩增片段大小符合预期的PCR产物回收纯化并进行测序。
(4)基因水平验证：将PCR产物测序，测序结果与野生型PCR序列比对，确定K3、K7-1和K7-2为突变体，序列分析如图1右侧d-f)所示。
(5)蛋白水平验证：在基因水平验证突变细胞株后，用蛋白质免疫印迹(Westernblotting)进一步验证K3、K7-1和K7-2是KDM2A缺陷型，结果见图2，未检测到KDM2A蛋白。
通过以上DNA水平以及蛋白水平的验证，证明KDM2A基因敲除的HEK293T细胞构建成功，然后使用K3表示14号外显子突变产生的2株敲除系，K7-1和K7-2表示18号外显子突变产生的2株敲除系。
实施例2：流式细胞仪检测KDM2A分别对野生型细胞系和敲除系的增殖和凋亡的影响
1、为了检测KDM2A基因的敲除会对细胞产生如何具体的影响，我们对野生型HEK293T细胞和敲除系分别进行了EdU细胞增殖检测和Annexin V-FITC/PI细胞凋亡检测。操作流程如下：
(1)将六孔板中每孔接种1×105～3×106个细胞，培养至正常阶段；
(2)按说明书配制EdU培养基，设置一个不加EdU培养基的空白对照，以便进行流式检测数据的染料背景分析。将细胞培养基更换为EdU培养基和对照培养基后，每孔加入1mL，孵育2小时；
(3)将细胞转移至1.5mL EP管中，收集细胞弃上清，再用PBS重悬细胞，离心弃上清；
(4)按EdU细胞增殖检测说明和Annexin V-FITC/PI凋亡检测说明进行染色，之后在相应的波长下进行流式分析及荧光显微成像。以上实验独立进行了3次重复，结果见图3、图4和图5。
2、通过流式细胞分析野生型细胞和敲除型细胞在细胞增殖和凋亡中的差异，发现KDM2A本身能够促进细胞增殖并且兼具降低细胞凋亡的功能。
虽然，本文中已经用一般性说明及具体实施方案对本发明做出了详尽的描述，但本领域技术人员可以在本发明基础上对其做一些修改或改进。因此在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 中南民族大学
<120> 利用CRISPR-CAS9技术敲除HEK293T细胞KDM2A基因的方法
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
caccgacatc gcactcgtct ccgtc 25
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
caccgtttga agcgctcatc gcggc 25
<210> 3
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aaacgacgga gacgagtgcg atgtc 25
<210> 4
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aaacgccgcg atgagcgctt caaac 25
<210> 5
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tgtaatcttc taccctatcc a 21
<210> 6
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aagaggagga ctcaacca 18
<210> 7
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tgtcggttca gagggtttg 19
<210> 8
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ccacaggctt tcagatgc 18
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gagggcctat ttcccatgat tcc 23

Claims (8)

1.一种基于CRISPR/CAS9系统的特异性敲除人源KDM2A基因的sgRNA，其特征在于：所述sgRNA在KDM2A基因上的靶序列位于KDM2A基因(NM_012308.2)的14号和18号外显子上，其中14号外显子上的靶序列为：5’-ACATCGCACTCGTCTCCGTC-3’，18号外显子上的靶序列为：5’-TTTGAAGCGCTCATCGCGGC-3’。
2.权利要求1所述的sgRNA的表达载体pX458-KDM2A。
3.权利要求2所述表达载体pX458-KDM2A的构建方法，其特征在于：在14号外显子以及18号外显子上的靶序列的5’端加上BbsI的酶切位点caccg，依次构成序列SEQ ID NO：1和SEQ ID NO：2，并人工合成其互补序列SEQ ID NO：3和SEQ ID NO：4，将互补序列SEQ IDNO.1和SEQ ID NO：3，以及SEQ ID NO.2和SEQ ID NO：4所形成的双链DNA，与经过BbsI酶切的pX458载体连接，从而制得表达载体pX458-KDM2A。
4.权利要求2所述的表达载体pX458-KDM2A在制备KDM2A基因敲除的HEK293T细胞系中的应用。
5.一种KDM2A基因敲除的HEK293T细胞系。
6.权利要求5所述的KDM2A基因敲除的HEK293T细胞系的构建方法，其特征在于包括以下步骤：以表达载体pX458-KDM2A作为KDM2A基因的打靶载体，转染HEK293T细胞，获得KDM2A基因敲除的单克隆细胞株，分别简记为K3、K7-1和K7-2。
7.根据权利要求6所述的KDM2A基因敲除的HEK293T细胞系的构建方法，其特征在于具体的构建方法如下：
(1)、细胞转染：培养HEK293T细胞至70～90％汇合度时准备转染，将含有表达载体pX458-KDM2A的混合液加入HEK293T细胞中进行转染，然后培养；
(2)、细胞基因组提取：将转染后的HEK293T细胞进行基因组提取；
(3)、PCR鉴定：根据KDM2A基因序列，设计引物KDM2A-3F和KDM2A-3R、KDM2A-7F和KDM2A-7R，上述引物的序列分别如：SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示；利用上述引物对所提取的基因组进行PCR反应；
(4)、基因水平验证：将PCR产物测序，测序结果与野生型PCR序列比对，确定K3、K7-1和K7-2为突变体；
(5)、蛋白水平验证：在基因水平验证突变细胞株后，用蛋白质免疫印迹进一步验证K3、K7-1和K7-2是KDM2A基因缺陷型。
8.权利要求5所述的KDM2A基因敲除的HEK293T细胞系在研究KDM2A蛋白的增殖和凋亡以及潜在的分子生物学机制研究中的应用。
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