CN114934068A - Alkbh5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用 - Google Patents
Alkbh5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用。本发明利用CRISPR/Cas 9相关技术,经过sgRNA的设计、插入片段的合成及酶切处理,构建出用于敲除ALKBH5的重组质粒,通过脂质体瞬转的方法,将质粒转到入小鼠卵巢颗粒细胞mGC中,经过嘌呤霉素稳转筛选以及单克隆的挑选及验证,从而建立了ALKBH5基因稳定敲除的细胞株ΔALKBH5‑mGC。本发明的细胞株可为研究ALKBH5对多囊卵巢综合症的致病机制的进一步深入研究搭建一个良好的基础。
Description
技术领域
本发明涉及一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用。
背景技术
多囊卵巢综合症(PCOS)作为最常见的异质性内分泌疾病之一,在全球育龄期妇女中的患病率约为6-13%,它不仅是女性无排卵性不孕的主要原因,还可增加糖尿病、血管血栓形成、子宫内膜异位症、子宫内膜癌和心血管疾病等多种疾病的风险。其诊断依据为高雄激素血症、卵巢多囊样改变和排卵功能障碍。尽管在过去的数十年间,对PCOS的治疗取得了较大的进展,但由于其高度的异质性,目前的治疗策略仍无法治愈这种疾病,因此,深入研究PCOS的病因势在必行。
卵泡是卵巢的核心功能单位,由单个卵母细胞和周围颗粒细胞、卵丘细胞和泡膜细胞组成。颗粒细胞作为卵泡内的体细胞,在卵泡发生、甾体激素生成以及生育能力的维持方面发挥了重要作用。越来越多的证据表明,颗粒细胞的功能障碍会导致卵泡对促性腺激素的反应能力、卵母细胞发育等的异常,这些失调都与PCOS的发病机制密切相关。多个研究表明,在PCOS患者的颗粒细胞中发现了异常的基因表达谱,多个差异表达基因与PCOS的发病机制密切相关,然而这些基因的调控机制在很大程度上仍不清楚。申请人前期通过对从PCOS患者颗粒细胞中提取的RNA进行RNA-seq测序,对测序结果进行GO富集分析等生信分析后,我们发现,PCOS患者中m6A去甲基化酶ALKBH5的mRNA水平显著下降。
m6A是真核细胞中最常见的修饰形式。它在不改变碱基序列的条件下,能够调控RNA的转录、剪接、降解和翻译过程,并且这种修饰是动态的、可逆的,由三种不同类型的蛋白复合物调节,分别为m6A甲基转移酶(Writers)、m6A甲基化阅读蛋白(Readers)以及m6A去甲基酶(Erasers)。简而言之,m6A修饰通过m6A甲基转移酶和m6A去甲基酶来对RNA进行甲基化修饰或去甲基化,而m6A甲基化阅读蛋白则用来识别m6A修饰。随着高通量测序技术在检测m6A方面的应用,研究发现m6A修饰在终止密码子、3‘UTR区附近富集,并在5’UTR区附近以不依赖帽子结构的方式进行翻译。目前的研究表明,m6A甲基化水平与多种疾病的发生发展有关,例如肿瘤、阿尔茨海默病、牙周炎、心力衰竭、人腹主动脉瘤和肥胖症等。
ALKBH5作为一种重要的m6A去甲基化酶,在对RNA的m6A的修饰调控上发挥了重要作用。研究人员已经证明ALKBH5参与了细胞的增殖、迁移、侵袭、转移、肿瘤以及生殖系统疾病的发生发展。在生殖细胞中,ALKBH5作为主要的m6A修饰的“橡皮擦”,发挥着氧化逆转m6A的作用。研究人员发现,ALKBH5缺失的雄性小鼠不育,且其mRNA的m6A修饰增加,他们认为导致雄性小鼠不育的原因是由于m6A过度积累导致RNA的异常剪接,产生较短的转录本,导致精子减数分裂受阻。而ALKBH5在颗粒细胞中如何发挥作用,以及ALKBH5的缺失是否与多囊卵巢综合症的发生相关,目前仍不明确。
现有技术中,通过siRNA方法敲降ALKBH5基因,在研究中需要多次转染,这无疑增加了人力投入和研究成本,且会存在敲降效率不高及不稳定的现象。
因此,亟需一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株的构建方法。
发明内容
本发明的目的是针对现有技术的不足,提供一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株及其构建方法和应用,该细胞株可为研究ALKBH5对多囊卵巢综合症的致病机制的进一步深入研究搭建一个良好的基础。
本发明的目的是通过以下技术方案来实现的:本发明实施例的第一方面提供了一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株的构建方法,包括以下步骤:
(1)设计一对互补的靶向小鼠ALKBH5基因第一外显子的sgRNA,以PX459 V2.0质粒为载体,构建出一个重组质粒,所述sgRNA的序列包括如SEQ ID NO.1所示的正向引物ALKBH5-sgRNA F序列,和如SEQ ID NO.2所示的反向引物ALKBH5-sgRNA R序列;
(2)将所述重组质粒转染进经永生化处理的小鼠卵巢颗粒细胞中,使用嘌呤霉素进行阳性细胞筛选,分离单克隆细胞进行培养,获得ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株。
进一步地,所述步骤1中,所述以PX459 V2.0质粒为载体,构建出一个重组质粒包括如下子步骤:
(1.1)将两条所述sgRNA退火形成双链,经T4激酶磷酸化;
(1.2)使用Bbs I限制性核酸内切酶切割位于PX459 V2.0质粒第245和267个碱基上的Bbs I酶切位点,将质粒切割成线性化;然后利用SanPrep柱式PCR产物纯化试剂盒纯化回收DNA片段;
(1.3)将步骤(1.1)的产物与与步骤(1.2)回收的DNA片段利用T4 DNA连接酶进行连接,并转化至DH5α感受态细胞中,接种至带有氨苄抗性的平板上,37℃培养12-16h后,挑取单克隆菌落;
(1.4)用U6启动子序列通用引物进行测序,提取测序正确的质粒。
进一步地,所述步骤(1.1)中,两条sgRNA退火形成双链的过程具体为:将样品在37℃条件下孵育30min后,在95℃条件下处理2min,随后从50℃开始,以每秒下降0.1℃的速度进行退火,直到体系温度降至20℃,最后将样品放至4℃保存。
进一步地,所述步骤(2)具体包括以下子步骤:
(2.1)将经永生化处理的小鼠卵巢颗粒细胞接种到12孔板培养12-16小时,再将转染试剂和重组质粒按3uL:1ug的比例混匀后的转染复合物滴入孔中,轻摇细胞培养板使其分布均匀后,放入培养箱培养48小时;
(2.2)往细胞培养板中加入含5ug/mL嘌呤霉素的培养基,培养24h后更换含有同样浓度嘌呤霉素的培养基继续培养,后每48h更换一次含5ug/mL嘌呤霉素的新鲜培养基,直到转染组细胞长满培养皿并且几乎没有死亡脱落的细胞。
(2.3)将细胞消化制备细胞悬液,倍比稀释后接种至96孔板,使各孔含有0.5-1个细胞。隔日细胞贴壁后,观察单克隆的个数,并标记。待单克隆密度长到孔的50-60%时,胰酶消化细胞,获得ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株。
本发明实施例的第二方面提供了一种由上述的构建方法构建得到的ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株。
本发明实施例的第三方面提供了一种上述的ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株作为ALKBH5对多囊卵巢综合症的致病机制研究的细胞模型的应用。
本发明具有如下有益效果:本发明利用PX459 V2.0质粒载体构建的PX459 V2.0-ALKBH5携带有CRISPR/Cas9系统的Cas核酸内切酶基因;CRISPR/Cas基因编辑技术成功永久性的敲除了mGC细胞ALKBH5基因,获得了ALKBH5敲除的稳定细胞株,为研究ALKBH5对多囊卵巢综合症的致病机制的进一步深入研究搭建一个良好的基础。
附图说明
图1为本发明构建的含有敲除基因使用的引导sgRNA的重组质粒模式图;
图2为本发明提供的ALKBH5敲除的稳定mGC细胞株Alkbh5蛋白表达检测结果图;
图3为本发明提供的ALKBH5敲除的稳定mGC细胞株细胞自噬相关蛋白表达检测结果图。
具体实施方式
下面结合附图,对本发明进行详细说明。在不冲突的情况下,下述的实施例及实施方式中的特征可以相互组合。
实施例1:载体构建
本发明方法利用CRISPR/Cas 9相关技术,经过sgRNA的设计、插入片段的合成及酶切处理,构建出用于敲除ALKBH5的重组质粒,具体包括以下子步骤:
1)ALKBH5基因的sgRNA设计
设计一对互补的靶向小鼠ALKBH5基因第一外显子的sgRNA的过程具体为:sgRNAoligo序列的设计应用https://benchling.com网站设计一对针对ALKBH5 cds区的外显子的sgRNA序列。设计方法如下:利用该网站提供的基因数据库,选择我们所需要的目的基因,将其导入,在网站提供的序列结果中,选择on-target得分高,off-target得分高的序列,在其两端加上酶切位点。在每条sgRNA序列F链的5‘端添加CACCAAG,R链的5’端添加AAAC,3‘端添加C,与PX459 V2.0质粒经Bbs I酶切后形成的黏性末端互补。如F链的5‘端第一个碱基是G,则添加碱基时末尾不需要补G,相应的R链3’端也不需要补C。以下将PX459 V2.0重组质粒称为PX459 V2.0-ΔALKBH5。
表1:sgRNA寡核苷酸序列
2)将两条所述sgRNA退火形成双链,经T4激酶磷酸化
将两条sgRNA寡聚核苷酸单链退火形成双链,经T4激酶磷酸化,退火反应体系包括浓度为100uM的ALKBH5-sgRNA F和ALKBH5-sgRNA R所示引物各1uL、10*NEB buffer2 1uL、T4-PNK0.5uL、ddH2O6.5uL,放置PCR仪中反应,反应程序为37℃反应30min,95℃反应2min,再以0.1℃/s的速率,降温至20℃,即获得sgRNA的双链DNA片段,放4℃冰箱保存,准备与酶切载体连接。
3)载体酶切并回收
利用Bbs I限制性核酸内切酶将PX459 V2.0质粒进行线性化,然后用SanPrep柱式PCR产物纯化试剂盒(上海生工生物)纯化酶切产物。
具体步骤为:取PX459 V2.0载体1ug,用Bbs1酶切,酶切体系为:PX459 V2.0 1ug、Bbs1 1uL、10*NEB cut smart buffer 5ul,加ddH2O补足体系到50uL。混匀后37℃水浴1小时。使用酶切产物回收试剂盒对酶切产物进行回收纯化。
4)sgRNADNA片段与PX459 V2.0载体的DNA连接
酶连体系:2uL 10*T4buffer、1uL T4酶、2uL载体、10uL sgRNADNA片段,加ddH2O补足体系到20uL;混匀后于37℃水浴反应4小时。
5)连接产物转化及菌落PCR鉴定,构建出一个重组质粒
取上述连接产物5uL加入到50uL DH5α感受态细胞中,先冰浴30min,42℃水浴45s后冰上放置2min,加入到500uL不含抗生素的培养基中37℃摇床震荡45-60min;将菌液涂到氨苄抗性的平板上,37℃培养箱培养12-16h;挑取单克隆菌落进行菌落扩增,用U6启动子序列通用引物进行测序,鉴定正确的重组质粒PX459 V2.0-ALKBH5-sgRNA(如图1所示)进行菌液抽提重组质粒并保存。
实施例2:制备ALKBH5基因敲除小鼠卵巢颗粒细胞株
1)实验分组
分为阳性组(PX459 V2.0-ALKBH5-sgRNA质粒+转染试剂),阴性对照组(阴性对照质粒+转染试剂),空白对照组(转染试剂);
2)细胞转染
本发明选用的小鼠卵巢颗粒细胞是由本实验室通过提取原代小鼠卵巢颗粒细胞后,将带有SV70基因片段的质粒载体转染进原代小鼠卵巢颗粒细胞,通过嘌呤霉素药物筛选的方法,得到抗性单克隆,扩大培养后建成小鼠卵巢颗粒细胞。随后,将mGC细胞以1.5*105/孔的密度接种到12孔板中,每孔含完全培养基1mL,每组设3组重复,放置细胞培养箱培养12-16小时,将孔内培养基更换为新鲜完全血清培养基。将37.5uL转染试剂lipofectamine3000(ThermoFisher Scientific)与12.5ug质粒分别逐滴加入到5mL不含血清的培养基的灭菌离心管中,混匀后室温孵育5min,再将混匀的转染试剂分别与PX459V2.0-ALKBH5-sgRNA质粒以及阴性对照质粒混合,室温孵育20min,每次取1mL转染复合物逐一滴入孔中。前后轻摇细胞培养板,使转染复合物在培养基中分布均匀。继续放入培养箱中培养,6小时更换新鲜完全培养基。
3)嘌呤霉素筛选
在培养48h后,往实验组和对照组细胞中分别加入含有终浓度5ug/mL嘌呤霉素的新鲜培养基;培养24h后更换含有同样浓度嘌呤霉素的新鲜完全培养基继续培养,之后每48h更换一次含5ug/mL嘌呤霉素的新鲜培养基,直到未转染组的细胞完全死亡,转染组细胞长满培养皿并且几乎没有死亡脱落的细胞。
4)细胞单克隆的筛选
将细胞消化后制备细胞悬液,细胞计数后用含有5ug/mL嘌呤霉素的新鲜培养基倍比稀释细胞悬液。在96孔板中,每孔加入100uL细胞悬液,使各孔含有0.5-1个细胞。隔日细胞贴壁后,观察单克隆的个数,并标记。待单克隆密度长到孔的50-60%时,用胰酶消化细胞,取50%细胞提取细胞基因组进行测序验证,剩下50%细胞继续在96孔板中培养扩增。
5)Western Blot鉴定Alkbh5蛋白表达量的降低情况
使用细胞裂解液裂解稳定敲除细胞株后,Pierce Rapid Gold BCA试剂盒测定蛋白样品浓度。蛋白样品经SDS-PAGE分离后转移到PVDF膜上,用含有5%脱脂奶粉的TBS将PVDF膜在室温下封闭1h,然后在4℃下与相应的一抗孵育过夜。次日,用TBST清洗膜1h,然后在相应的以辣根过氧化物酶结合的二抗中孵育30min。同样,在二抗体孵育后用TBST洗膜1h。最后用增强型化学发光底物和X射线胶片检测免疫反应条带。使用Image-Pro Plus软件对条带的强度进行量化。
如图2所示,通过免疫印迹分析,我们发现ΔALKBH5-mGC的Alkbh5蛋白与对照组相比明显降低,以此确定了ΔALKBH5-mGC细胞株中的Alkbh5蛋白被成功敲除。
实施例3:ALKBH5基因稳定敲除小鼠卵巢颗粒细胞株的应用
1)Real time RT-qPCR
①利用TRIzol试剂(Takara,日本)按说明提取总RNA后,用PrimeScript RT试剂盒(Takara)将3μg RNA逆转录成互补DNA(CDNA)。
②20μL反应体系
实时定量PCR 20μL反应体系如下:
③基因定量
以GAPD(3-磷酸甘油醛脱氢酶)作为内参,每个cDNA样本的重复3次,取平均值。目的基因mRNA的相对浓度用2-ΔDelta;Ct公式进行计算。
通过RT-qPCR分析,我们发现ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株中,与PCOS相关的基因Ptx3、Star的mRNA水平较对照组显著降低,而Has2的mRNA水平较对照组显著升高,同时,我们还发现自噬相关基因Bnip3的mRNA水平在ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株中显著升高。
2)Western Blot鉴定自噬相关蛋白表达量的变化情况
具体实验步骤同实施例2中的5)Western Blot鉴定Alkbh5蛋白表达量的降低情况。
由于有研究发现,自噬参与了PCOS相关的代谢紊乱,并且我们RT-qPCR的结果发现自噬相关基因Bnip3表达量在ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株中显著升高,因此,我们推测,Alkbh5表达的降低可能是通过自噬来影响PCOS的发生。
如图3所示,我们发现ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株中,自噬相关蛋白LC3-I/II、Pink1较对照组显著升高。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明保护的范围之内。
序列表
<110> 浙江大学医学院附属妇产科医院
<120> 一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
caccgcacca agcggaaata ccagg 25
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aaaccctggt atttccgctt ggtgc 25
Claims (6)
1.一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株的构建方法,其特征在于,包括以下步骤:
(1)设计一对互补的靶向小鼠ALKBH5基因第一外显子的sgRNA,以PX459 V2.0质粒为载体,构建出一个重组质粒,所述sgRNA的序列包括如SEQ ID NO.1所示的正向引物ALKBH5-sgRNA F序列,和如SEQ ID NO.2所示的反向引物ALKBH5-sgRNA R序列;
(2)将所述重组质粒转染进经永生化处理的小鼠卵巢颗粒细胞中,使用嘌呤霉素进行阳性细胞筛选,分离单克隆细胞进行培养,获得ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株。
2.根据权利要求1所述的构建方法,其特征在于,所述步骤1中,所述以PX459 V2.0质粒为载体,构建出一个重组质粒包括如下子步骤:
(1.1)将两条所述sgRNA退火形成双链,经T4激酶磷酸化;
(1.2)使用Bbs I限制性核酸内切酶切割位于PX459 V2.0质粒第245和267个碱基上的Bbs I酶切位点,将质粒切割成线性化;然后利用SanPrep柱式PCR产物纯化试剂盒纯化回收DNA片段;
(1.3)将步骤(1.1)的产物与与步骤(1.2)回收的DNA片段利用T4 DNA连接酶进行连接,并转化至DH5α感受态细胞中,接种至带有氨苄抗性的平板上,37℃培养12-16h后,挑取单克隆菌落;
(1.4)用U6启动子序列通用引物进行测序,提取测序正确的质粒。
3.根据权利要求2所述的构建方法,其特征在于,所述步骤(1.1)中,两条sgRNA退火形成双链的过程具体为:将样品在37℃条件下孵育30min后,在95℃条件下处理2min,随后从50℃开始,以每秒下降0.1℃的速度进行退火,直到体系温度降至20℃,最后将样品放至4℃保存。
4.根据权利要求1所述的构建方法,其特征在于,所述步骤(2)具体包括以下子步骤:
(2.1)将经永生化处理的小鼠卵巢颗粒细胞接种到12孔板培养12-16小时,再将转染试剂和重组质粒按3uL:1ug的比例混匀后的转染复合物滴入孔中,轻摇细胞培养板使其分布均匀后,放入培养箱培养48小时;
(2.2)往细胞培养板中加入含5ug/mL嘌呤霉素的培养基,培养24h后更换含有同样浓度嘌呤霉素的培养基继续培养,后每48h更换一次含5ug/mL嘌呤霉素的新鲜培养基,直到转染组细胞长满培养皿并且几乎没有死亡脱落的细胞。
(2.3)将细胞消化制备细胞悬液,倍比稀释后接种至96孔板,使各孔含有0.5-1个细胞。隔日细胞贴壁后,观察单克隆的个数,并标记。待单克隆密度长到孔的50-60%时,胰酶消化细胞,获得ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株。
5.一种权利要求1-4中任一权利要求所述的构建方法构建得到的ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株。
6.一种权利要求要求5所述的ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株作为ALKBH5对多囊卵巢综合症的致病机制研究的细胞模型的应用。
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