CN116970647A - 一种wsb2基因敲除单克隆细胞株的构建方法及其应用 - Google Patents
一种wsb2基因敲除单克隆细胞株的构建方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种WSB2基因敲除单克隆细胞株的构建方法及其应用。该方法是采用CRISPR/Cas9系统来制备WSB2基因敲除单克隆细胞株,首先针对WSB2基因设计两个sgRNA序列,利用分子克隆技术构建重组质粒;然后将重组质粒转染至293T细胞进行病毒包装,收集的病毒液转染MDA‑MB‑231细胞,进行嘌呤霉素药物筛选和单克隆化处理,然后提取单克隆细胞株基因组DNA,进行WSB2基因水平测序鉴定,获得WSB2基因敲除MDA‑MB‑231细胞系。本发明构建的WSB2基因敲除MDA‑MB‑231细胞系是一种基因稳定遗传的细胞系,采用的方法简便、高效、快速、低成本。
Description
技术领域
本发明属于基因工程领域,涉及一种WSB2基因敲除单克隆MDA-MB-231细胞株的构建方法及其应用。
背景技术
WSB2(WD repeat and SOCS box-containing protein 2)属于WD蛋白亚家族,可能充当某些底物之间的桥结合域和E3泛素蛋白连接酶。WSB-2可以与IL-21R的近膜胞浆内区域结合,从而负调控IL-21R表达和IL-21诱导的信号转导(1);WSB-2与G-CSF-R的COOH末端区域结合,可能会导致髓样白血病(2);研究还表明WSB2敲低可以显著抑制了A375和G361细胞的周期进程和迁移,并且降低了c-Myc、β-catenin、CDK4和Cyclin D的蛋白水平(3)。WSB2 是miR-28-5p的靶基因,并且miR-28-5p参与调控乳腺癌的转移(4)。目前,未见有稳定的WSB2敲除细胞株的建立方法报导。为了深入研究WSB2的功能,亟需提供一种稳定且易于实施的WSB2敲除细胞株构建方法。
参考文献:
(1). Nara H, Onoda T, Rahman M, et al. Regulation of interleukin-21receptor expression and its signal transduction by WSB-2. Biochem Biophys ResCommun 392: 171-177, 2010.
(2). Erkeland SJ, Aarts LH, Irandoust M, et al. Novel role of WD40and SOCS box protein-2 in steady-state distribution of granulocyte colony-stimulating factor receptor and G-CSF-controlled proliferation anddifferentiation signaling. Oncogene 26: 1985-1994, 2007.
(3). Zhang Y, Li Z, Zhao W, et al. WD repeat and SOCS box containingprotein 2 in the proliferation, cycle progression, and migration of melanomacells. Biomed Pharmacother 116: 108974, 2019.
(4) . Ma L, Zhang Y and Hu F: miR28-5p inhibits the migration ofbreast cancer by regulating WSB2. Int J Mol Med 46: 1562-1570, 2020.
发明内容
本发明的目的是针对现有技术中尚未有稳定的WSB2敲除细胞株构建方法这一问题,提供一种构建WSB2基因敲除单克隆细胞株的方法及应用。所述方法是采用CRISPR/Cas9系统来制备WSB2基因敲除单克隆细胞株,首先针对WSB2基因设计两个sgRNA序列,利用分子克隆技术构建重组质粒;然后将重组质粒转染至293T细胞进行病毒包装,收集的病毒液转染MDA-MB-231细胞,进行嘌呤霉素药物筛选和单克隆化处理,然后提取单克隆细胞株基因组DNA,进行WSB2基因水平测序鉴定,获得WSB2基因敲除MDA-MB-231细胞系。本发明构建的WSB2基因敲除MDA-MB-231细胞系是一种基因稳定遗传的细胞系。
本发明一方面提供了一种WSB2基因敲除单克隆细胞株的构建方法,基于CRISPR/Cas9系统,
其中sgRNA识别序列包括:
sgRNA1:acgttaattcggaagctaga,
sgRNA2:tagcttccgaattaacgtgt;
所述方法包括如下步骤:
1)以PX548质粒为载体,分别构建含有sgRNA1的重组质粒PX458-WSB2-sgRNA1和含有sgRNA2的重组质粒PX458-WSB2-sgRNA2;
2)将重组质粒转染至293T细胞进行病毒包装,分别得到包装重组质粒PX458-WSB2-sgRNA1和PX458-WSB2-sgRNA2的病毒液;
3)将包装重组质粒PX458-WSB2-sgRNA1和PX458-WSB2-sgRNA2的病毒液共转染待敲除细胞,得到WSB2基因敲除单克隆细胞株。
在某些具体的实施方法中,所述步骤1)包括如下子步骤:
1.1)将sgRNA1的上下游引物或sgRNA2的上下游引物退火,经T4多聚核苷酸激酶磷酸化,得到sgRNA1退火产物和sgRNA2退火产物;
1.2)用BbSI限制性内切酶切割PX458质粒得到PX458酶切载体产物;
1.3)用T7 DNA连接酶将退火产物与PX458酶切载体产物连接,分别得到sgRNA1连接产物和sgRNA2连接产物;
1.4)将连接产物转化至stbl3大肠杆菌感受态细胞中,挑取单克隆菌落,培养后提取所述重组质粒PX458-WSB2-sgRNA1或PX458-WSB2-sgRNA2。
在优选的具体实施方法中,所述步骤1.1)中sgRNA1和sgRNA2的上下游引物分别为:
sgRNA1-上游引物(5’-3’),CACCGacgttaattcggaagctaga;
sgRNA1-下游引物(5’-3’),AAACtctagcttccgaattaacgtC;
sgRNA2-上游引物(5’ -3’),CACCGtagcttccgaattaacgtgt ;
sgRNA2-下游引物(5’-3’),AAACacacgttaattcggaagctaC ;
在某些具体的实施方法中,所述步骤2)包括如下子步骤:
2.1) 将293T细胞以5×105/孔接种于6孔板,培养24h后,弃培养基,PBS洗2次,添加1 ml Opti-MEM培养基;
2.2) 配制质粒混合液,其中含有: 12 µg/ml的重组质粒PX458-WSB2-sgRNA1或PX458-WSB2-sgRNA2; 8µg/ml的psPAX质粒; 3 µg/ml的pMD2.G质粒;溶剂为Opti-MEM;
2.3)配置聚乙烯亚胺溶液,其中聚乙烯亚胺的体积含量为40 µL/ml,溶剂为Opti-MEM;
2.4)将质粒混合液与聚乙烯亚胺溶液以1:1的体积比混合,室温静置孵育15 min;
2.5)将步骤2.4)所得混合液以1:1的体积比添加至步骤2.1)的培养基中继续培养18 h后吸去培养基,加入5 mL新配制的完全培养基;48 h后收集培养上清,离心,0.45 µm滤膜过滤,获得含有包装重组质粒PX458-WSB2-sgRNA1或PX458-WSB2sgRNA2的病毒液。
在某些具体的实施方法中,步骤3)包括如下子步骤:
1)将所述待敲除细胞以3×105/孔接种于6孔板,培养24 h;
2)在六孔板中加入含有包装重组质粒PX458-WSB2-sgRNA1和PX458-WSB2sgRNA2的病毒液各500 µL,摇匀;
3)细胞于培养箱中静置培养48 h。
在某些具体的实施方法中,所述待敲除细胞选自为MDA-MB-231、T47D、MCF-7、skov3、HepG2等人源细胞的一种,优选MDA-MB-231。
在某些具体的实施方法中,还包括用嘌呤霉素对所述WSB2基因敲除单克隆细胞株进行筛选,分离单克隆细胞进行培养,得到稳定的WSB2基因敲除单克隆细胞株。
本发明第二方面也提供了一种上述方法构建的WSB2基因敲除单克隆细胞株。
本发明第三方面也提供了一种WSB2基因敲除单克隆MDA-MB-231细胞株,保藏于中国典型培养物保藏中心,保藏地址:中国.武汉.武汉大学,保藏编号为CCTCC NO:C2023223;保藏日期:2023-07-19;分类命名:WSB2-基因敲除单克隆MDA-MB-231细胞株。
本发明还提供了一种上述的细胞株的应用,包括:作为对乳腺癌或者其他疾病致病机制研究的工具细胞;用于筛选或评估治疗肿瘤和其它相关疾病中的药物。
本发明具有以下有益效果:
本发明重点在于构建了两个用于定向敲除WSB2的sgRNA序列。设计好序列后,本发明采用CRISPR/Cas9系统构建得到了稳定的WSB2基因敲除单克隆细胞株。本发明提供的方法可用于WSB2基因的定向敲除致使其功能失活,具有简便、高效、快速、低成本等特点,对于WSB2基因功能和相关通路的研究具有重要意义。且本发明发现WSB2敲除后,相较于野生型细胞,细胞的增殖和迁移均受到了抑制。
附图说明
图1为sgRNA在WSB2基因的位点;
图2为 测序结果与WSB2基因序列的对比;
图3为 MDA-MB-231-KO-WSB2中氨基酸序列发生了移码突变;
图4为 MDA-MB-231-KO-WSB2细胞中WSB2的表达;
图5为 CCK-8检测细胞增殖;
图6为 Edu检测细胞S期细胞数目占比;
图7为 细胞克隆形成能力检测;
图8为 划痕实验检测伤口愈合情况;
图9为 迁移实验检测细胞迁移情况。
具体实施方式
以下将通过实施例来详细说明本申请的实施方式,借此对本申请如何应用技术手段来解决技术问题并达成技术功效的实现过程能充分理解并据以实施。
本申请中所用原料、设备,若无特别说明,均为本领域的常用原料、设备,均来自市售产品。本申请中所用方法,若无特别说明,均为本领域的常规方法。
本申请还存在其它多种可实施的技术方案,在此不做一一列举,本申请权利要求中要求保护的技术方案都是可以实施的。
“包含”或“包括”旨在表示组合物(例如介质)和方法包括所列举的要素,但不排除其他要素。当用于定义组合物和方法时,“基本上由……组成”意味着排除对于所述目的的组合具有任何重要意义的其他要素。因此,基本上由本文定义的元素组成的组合物不排除不会实质上影响要求保护的本申请的基本和新颖特征的其他材料或步骤。“由……组成”是指排除其他组成部分的微量元素和实质性的方法步骤。由这些过渡术语中的每一个定义的实施方案都在本申请的范围内。
实施例1
细胞培养:人类乳腺癌细胞系MDA-MB-231(中科院细胞库,TCHu227)和MDA-MB-231-KO-WSB2细胞培养在L-15培养基中;293T细胞(中科院细胞库,GNHu17)培养在DMEM培养基中;培养基中分别添加10%胎牛血清和双抗生素(100U/ml青霉素和100µg/ml链霉素);并将细胞置于37℃、5% CO2细胞培养箱中培养。
WSB2的序列如详见Entrez 数据库, Entrez ID:55884。
MDA-MB-231-KO-WSB2的构建
1.根据WSB2基因序列信息,在ORF的近5’端设计2个sgRNA(见图1):
sgRNA1,acgttaattcggaagctaga;
sgRNA2,tagcttccgaattaacgtgt。
根据sgRNAs及pSpCas9(BB)-2A-GFP (PX458)载体(北京启研生物科技有限司,Cat.NoQYV0273)特点设计克隆引物如下:
sgRNA1-上游引物(5’-3’),CACCGacgttaattcggaagctaga;
sgRNA1-下游引物(5’-3’),AAACtctagcttccgaattaacgtC;
sgRNA2-上游引物(5’ -3’),CACCGtagcttccgaattaacgtgt ;
sgRNA2-下游引物(5’-3’),AAACacacgttaattcggaagctaC 。
注:分别在上游引物5'端添加CACCG;下游引物5'端添加AAAC,3'端添加C。
引物由华大基因科技有限公司合成,上下游引物均采用ddH2O溶解为100 μM的溶液。
2.引物退火
在200μL EP管中分别配制总体积为10μL的如下退火反应体系:
退火反应体系1: sgRNA1-上下游引物各1 μL;T4多聚核苷酸激酶(纽英伦生物技术有限公司,M0201S)1μL;T4 多聚核苷酸激酶 buffer (纽英伦生物技术有限公司,B0201S)1 μL;ATP(10mM) 1 μL;ddH2O 5 μL。
退火反应体系2:sgRNA2-上下游引物各1μL;T4多聚核苷酸激酶1μL;T4 多聚核苷酸激酶 buffer 1 μL;ATP(10mM)1 μL;ddH2O 5 μL。
将退火反应体系1和退火反应体系2进行如下反应:37℃,30min;95℃,-2℃/sec;85℃,-0.5℃/sec;16℃保存。分别获得退火产物1和退火产物2。
3. PX458载体酶切
在200μL EP管中配制总体积为50μL的PX458载体酶切体系:PX458载体,1 ug;BbSI限制性内切酶(纽英伦生物技术有限公司,R3539S),1 ul; rCutSmart™ Buffer (纽英伦生物技术有限公司,B6004S ,5 ul;Quick CIP碱性磷酸酶(纽英伦生物技术有限公司,M0525L),1 ul;ddH2O至50 μL,吹打混匀。
上述酶切体系在如下条件进行反应:冰上大于1小时;37℃,10 min;80℃,2 min。
按照PCR产物纯化试剂盒(生工生物工程股份有限公司,B518141)说明书要求回收上述酶切产物,用50 μL洗脱液洗脱,获得PX458酶切载体产物。
4.连接
在200μL EP管中配制总体积20μL的连接反应体系:
连接反应体系1:步骤3回收的PX458酶切载体产物,50ng;步骤2获得退火产物1,1μL;T7 DNA连接酶Buffer(纽英伦生物技术有限公司,M0318S),10μL;T7 DNA连接酶(纽英伦生物技术有限公司,M0318S),1μL;ddH2O至20μL。
连接反应体系2:步骤3回收的PX458酶切载体产物,50ng;步骤2获得退火产物2,1μL;T7 DNA连接酶Buffer,10μL;T7 DNA连接酶,1μL;ddH2O至20μL。
上述连接反应体系1和连接反应体系2在如下条件反应:25℃,30min或4℃,过夜。分别获得连接产物1和连接产物2。
5.转化、测序、质粒提取
1)将步骤4获得的5 μL的连接产物1或连接产物2分别转化到stbl3大肠杆菌感受态细胞中,涂板含有Amp的LB平板培养,37℃,12-16h;
2)挑取2个单克隆菌落,于含有Amp的LB培养基中扩增菌体;
3)送菌液测序。测序结果sgRNA序列部分完全正确;
4)按照无内毒素质粒提取试剂盒(天根生化科技有限公司,DP118)的说明书要求,提取质粒PX458-WSB2-sgRNA1或 PX458-WSB2-sgRNA2。
6.病毒包装
1) 培养293T细胞,使其处于生长旺盛状态,以5×105/孔接种于6孔板。
2) 培养24h后,弃培养基,PBS洗2次,添加1 ml Opti-MEM(ThermoFisher,31985062)培养基。
3) 配制质粒混合液: PX458-WSB2-sgRNA1或 PX458-WSB2-sgRNA2,12 µg;psPAX质粒(北京启研生物科技有限司, QYV0052) 8 µg;pMD2.G质粒(北京启研生物科技有限司,QYV0053 ) 3 µg;添加Opti-MEM(ThermoFisher,31985062) 至1 ml
4) 配制PEI-Opti-MEM :聚乙烯亚胺( Sigma-Aldric,764604)40µL;添加Opti-MEM 至1 ml
5) 按照1:1体积比将PEI-Opti-MEM和上述质粒混合液混匀,室温静置孵育15min。
6) 将按照1:1体积比将上一步混合液滴加到培养基中,轻轻晃动摇匀,在培养箱中继续培养。
7) 18 h后吸去培养基,加入5 mL新配制的完全培养基(DMEM+10%胎牛血清+双抗生素(100U/ml青霉素和100µg/ml链霉素))。
48 h后收集培养上清,离心,0.45 µm滤膜过滤,分别获得PX458-WSB2-sgRNA1和PX458-WSB2sgRNA2病毒液,液氮速冻,-80℃保存。
7.细胞转染
1) 培养MDA-MB-231细胞,使其处于生长旺盛状态,以3×105/孔接种于6孔板,继续培养24 h。
2) 在六孔板中加入500 µL的PX458-WSB2-sgRNA1和PX458-WSB2sgRNA2病毒液,摇匀。
3) 细胞于培养箱中静置培养48 h。
8.阳性细胞多克隆筛选
1) 细胞转染48 h后,更换培养基(L15+10%胎牛血清+双抗生素(100U/ml青霉素和100µg/ml链霉素)+ 0.5 μg/mL嘌呤霉素),随后每48 h更换一次。
2) 筛选6 d后,对照组MDA-MB-231细胞全部死亡,将实验组阳性细胞扩大培养。
9.阳性单克隆细胞株筛选
1) 将阳性多克隆细胞培养至融合度约75-85%,使其处于生长;
2) 细胞计数,将细胞悬液稀释成5 个细胞/mL;
3) 混匀后96孔板每孔加100 μL细胞悬液,培养箱中培养。
4) 静置培养24 h后,更换培养基(L15+10%胎牛血清+双抗生素(100U/ml青霉素和100µg/ml链霉素)+ 0.5 μg/mL嘌呤霉素),每日观察并记录单克隆形成情况。
5) 挑选阳性单克隆进行扩增,测序验证。
10.基因敲除阳性单克隆细胞株验证——测序
1) 按照细胞基因组DNA提取试剂盒(生工生物工程股份有限公司,B518221)说明书要求,提取抗性筛选阳性的单克隆细胞基因组;
2) PCR扩增WSB2基因靶标序列
引物设计如下:
WSB2-1-上游引物(5’-3’),agaGGATCCtccttgagcatgcccattgtt(BamH I)
WSB2-1-下游引物(5’-3’),tatAAGCTTactggagtgacctcagcctt (Hind III)
注:GGATCC是BamH I酶切位点;AAGCTT是Hind III酶切位点。
PCR反应体系的配制: 2 x Phanta Master Mix(诺唯赞生物科技股份有限公司,P515) 10 µL;WSB2-1上下游引物(10 µM )各 0.5 µL;模板DNA 50ng;ddH2O 至 20µL。
反应程序:95°C 3 min; 95°C 15 sec,59℃ 15 sec, 72°C 28 sec,35个循环;72°C 5 min;保持 4°C。
3)按照常规方法 将目的基因片段克隆至pUC18载体(北京启研生物科技有限司,QYV0757),挑取单克隆菌落;
4) 送样测序验证阳性单克隆细胞株的WSB2基因靶标序列,以确定是否敲除成功。
11. Western blot 鉴定
对MDA-MB-231-WSB2-KO单克隆细胞和MDA-MB-231细胞进行培养,采用RIPA细胞裂解液(北京索莱宝科技有限公司,R0010)提取细胞总蛋白,western blot 检测敲除WSB2基因的效果,利用Protein Assay Kit(北京索莱宝科技有限公司,PC0020)对总蛋白进行定量,聚丙烯酰胺凝胶蛋白电泳,采用一抗anti-WSB2 (爱必信生物科技有限公司,abs110751),anti--actin (Abcam,ab8227)进行常规Western Blot分析。
实验结果:
MDA-MB-231-WSB2-KO单克隆细胞测序结果表明:在sgRNA靶标位点发生突变敲除1个碱基a(见图2)。因此在新的氨基酸序列中引起了移码突变和翻译提前终止(见图3)。确认该单克隆细胞株为纯合突变,扩增该细胞,并冻存保种。
western blot结果表明:MDA-MB-231-WSB2-KO单克隆细胞中WSB2蛋白表达缺失(见图4),表明已敲除MDA-MB-231细胞中的WSB2基因。
实施例2:MDA-MB-231-WSB2-KO细胞株细胞增殖和迁移能力的检测
1. CCK-8实验
将MDA-MB-231-WSB2-KO或MDA-MB-231细胞按5×103个细胞/孔接种到96孔细胞培养板中, 24小时后,每孔加入10µL CCK-8溶液。CO2培养箱培养1h后,酶标仪测量450 nm吸光度。
2.Edu增殖实验
将MDA-MB-231-WSB2-KO或MDA-MB-231细胞按5×103个细胞/孔接种到96孔细胞培养板中,在37℃ 、5% CO2细胞培养箱中孵育24h。按照锐博Edu Apollo 488试剂盒操作步骤进行细胞固定及染色,染色完成后在荧光显微镜下进行观察并拍照保存。
3.平板克隆形成实验
将MDA-MB-231-WSB2-KO或MDA-MB-231细胞按1.5×103个细胞接种到6孔板中,将其置于37℃、5% CO2细胞培养箱中培养7天。结晶紫染色,拍照记录克隆数量。
4.划痕实验
将MDA-MB-231-WSB2-KO或MDA-MB-231细胞按5×103个细胞/孔接种到96孔细胞培养板中,在37℃ 、5% CO2细胞培养箱中孵育24h。划痕后,PBS清洗3-5次,于0h、24h、48h和72h后拍照检测伤口愈合情况。
5.迁移实验
将MDA-MB-231-WSB2-KO或MDA-MB-231细胞按2×104个细胞接种到到Transwell小室中,并将小室放入24孔板,24孔板内Transwell小室下层加入0.75ml 含10%血清的细胞培养基,将其置于37℃、5% CO2细胞培养箱中培养16h。最后用棉签抹去Transwell小室上层残余细胞,下层细胞用0.25%的结晶紫于37℃染色10min,置于光学倒置显微镜下观察穿膜细胞数量并拍照记录。
实验结果:相较于MDA-MB-231细胞,MDA-MB-231-WSB2-KO的细胞数目(图5)及处于S期的细胞数目(图6)均显著降低,因此,敲除WSB2后,致使S期的数目减少,细胞增殖受到抑制。并且相较于MDA-MB-231细胞,MDA-MB-231-WSB2-KO的克隆形成能力(图7)、伤口愈合能力(图8)和迁移能力均显著减弱(图9)。
本申请说明书中未作详细描述的内容属于本领域技术人员的公知常识。
如在通篇说明书及权利要求当中所提及的“包含”为一开放式用语,故应解释成“包含但不限定于”。“大致”是指在可接收的误差范围内,本领域技术人员能够在一定误差范围内解决所述技术问题,基本达到所述技术效果。
还需要说明的是,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的商品或者系统不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种商品或者系统所固有的要素。在没有更多限制的情况下,由语句“包括一个……”限定的要素,并不排除在包括所述要素的商品或者系统中还存在另外的相同要素。
上述说明示出并描述了本申请的若干优选实施例,但如前所述,应当理解本申请并非局限于本文所披露的形式,不应看作是对其他实施例的排除,而可用于各种其他组合、修改和环境,并能够在本文所述发明构想范围内,通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离本申请的精神和范围,则都应在本申请所附权利要求的保护范围。
Claims (10)
1.一种WSB2基因敲除单克隆细胞株的构建方法,其特征在于,基于CRISPR/Cas9系统,
其中sgRNA识别序列包括:
sgRNA1:acgttaattcggaagctaga,
sgRNA2:tagcttccgaattaacgtgt;
所述方法包括如下步骤:
1)以PX548质粒为载体,分别构建含有sgRNA1的重组质粒PX458-WSB2-sgRNA1和含有sgRNA2的重组质粒PX458-WSB2-sgRNA2;
2)将重组质粒转染至293T细胞进行病毒包装,分别得到包装重组质粒PX458-WSB2-sgRNA1和PX458-WSB2-sgRNA2的病毒液;
3)将包装重组质粒PX458-WSB2-sgRNA1和PX458-WSB2-sgRNA2的病毒液共同转染待敲除细胞,得到WSB2基因敲除单克隆细胞株。
2.根据权利要求1所述的方法,其特征在于,所述步骤1)包括如下子步骤:
1.1)将sgRNA1的上下游引物或sgRNA2的上下游引物退火,经T4多聚核苷酸激酶磷酸化,得到sgRNA1退火产物和sgRNA2退火产物;
1.2)用BbSI限制性内切酶切割PX458质粒得到PX458酶切载体产物;
1.3)用T7 DNA连接酶将退火产物与PX458酶切载体产物连接,分别得到sgRNA1连接产物和sgRNA2连接产物;
1.4)将连接产物转化至stbl3大肠杆菌感受态细胞中,挑取单克隆菌落,培养后提取所述重组质粒PX458-WSB2-sgRNA1或PX458-WSB2-sgRNA2。
3.根据权利要求2所述的方法,其特征在于,所述步骤1.1)中sgRNA1和sgRNA2的上下游引物分别为:
sgRNA1-上游引物(5’-3’),CACCGacgttaattcggaagctaga;
sgRNA1-下游引物(5’-3’),AAACtctagcttccgaattaacgtC;
sgRNA2-上游引物(5’ -3’),CACCGtagcttccgaattaacgtgt;
sgRNA2-下游引物(5’-3’),AAACacacgttaattcggaagctaC。
4.根据权利要求1所述的方法,其特征在于,所述步骤2)包括如下子步骤:
2.1) 将293T细胞以5×105/孔接种于6孔板,培养24h后,弃培养基,PBS洗2次,添加1 mlOpti-MEM培养基;
2.2) 配制质粒混合液,其中含有: 12 µg/ml的重组质粒PX458-WSB2-sgRNA1或PX458-WSB2-sgRNA2; 8 µg/ml的psPAX质粒;3 µg/ml的pMD2.G质粒;溶剂为Opti-MEM;
2.3)配置聚乙烯亚胺溶液,其中聚乙烯亚胺的体积含量为40 µL/ml,溶剂为Opti-MEM;
2.4)将质粒混合液与聚乙烯亚胺溶液以1:1的体积比混合,室温静置孵育15 min;
2.5)将步骤2.4)所得混合液以1:1的体积比添加至步骤2.1)的培养基中继续培养18 h后吸去培养基,加入5 mL新配制的完全培养基;48 h后收集培养上清,离心,0.45 µm滤膜过滤,获得含有包装重组质粒PX458-WSB2-sgRNA1或PX458-WSB2sgRNA2的病毒液。
5.根据权利要求4所述的方法,其特征在于,步骤3)包括如下子步骤:
1)将所述待敲除细胞以3×105/孔接种于6孔板,培养24 h;
2)在六孔板中加入含有包装重组质粒PX458-WSB2-sgRNA1和PX458-WSB2sgRNA2的病毒液各500 µL,摇匀;
3)细胞于培养箱中静置培养48 h。
6.根据权利要求5所述的方法,其特征在于,所述待敲除细胞选自为MDA-MB-231、T47D、MCF-7、skov3、HepG2等人源细胞的一种,优选MDA-MB-231。
7.根据权利要求1所述的方法,其特征在于,还包括用嘌呤霉素对所述WSB2基因敲除单克隆细胞株进行筛选,分离单克隆细胞进行培养,得到稳定的WSB2基因敲除单克隆细胞株。
8.一种权利要求1-7任一所述方法构建的WSB2基因敲除单克隆细胞株。
9.一种WSB2基因敲除单克隆MDA-MB-231细胞株,其特征在于,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:C2023223。
10.一种权利要求8或9所述的细胞株的应用,其特征在于,包括:作为对乳腺癌或者其他疾病致病机制研究的工具细胞;用于筛选或评估治疗肿瘤和其它相关疾病中的药物。
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