CN106047803A - CRISPR/Cas9靶向敲除兔BMP2基因的细胞模型及其应用 - Google Patents
CRISPR/Cas9靶向敲除兔BMP2基因的细胞模型及其应用 Download PDFInfo
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Abstract
本发明属于分子生物学与生物医学技术领域,具体的说,本发明涉及基于CRISPR/Cas9靶向敲除兔骨形态发生蛋白2(BMP2)基因的细胞模型及其应用。本发明根据CRISPR/Cas9的设计原则,在兔BMP2基因上3个靶向位点,合成相应的oligos,并且将其构建在px458载体上。在兔骨髓间充质干细胞中利用针对这3个靶点构建出的CRISPR/Cas9系统,可以有效的敲除兔BMP2基因,这一系统易于操作,兔BMP2基因敲除效率高。本发明公开的兔BMP2基因敲除细胞模型能极大地促进BMP2基因功能及信号通路的相关研究。
Description
技术领域
本发明属于分子生物学与生物医学技术领域,具体涉及基于CRISPR/Cas9靶向敲除兔骨形态发生蛋白2(BMP2)基因的细胞模型及其应用。
背景技术
CRISPR/Cas系统是从细菌和古生菌对抗外来病毒或质粒的适应性免疫系统发展而来,包括三种不同的类型,其中TypeII型的CRISPR/Cas系统的DNA内切酶Cas9只有一个亚基,结构最为简单,所以应用也最广泛。除了Cas9蛋白外,该系统还包括两条短的CRISPRRNAs(crRNAs)和trans-activatingcrRNAs(tracrRNA)。成熟的crRNA-tracrRNA复合体可以通过碱基互补配对指导Cas9蛋白到靶序列上,并在PAM(protospaceradjacentmotif)附近特异性剪切DNA双链,形成DSB(doublestrandbreak)。DSB可以通过两种途径被修复,一种是非同源重组的末端接合(Non-HomologousEndJoiningNHEJ)DNA修复方式,另一种是同源重组修复(HomologyDirectedRepairHDR)方式。NHEJ修复方式可能产生碱基的插入或缺失,从而产生移码突变,或者也可能突变成终止密码子,这些突变形式都可以改变目的基因的开放阅读框;HDR方式需要一段与被剪切片段同源的模板片段来修复DSB,这种修复方式可以将被用来作为模板的同源片段的序列复制到目的基因中,所以可以利用这种修复方式将特定的基因片段引入到目的基因中。
骨形态发生蛋白(bone morphogenetic proteins,BMPs)又名成骨蛋白(osteogenicproteins,OPs),属于转化生长因子β(transforming growth factor-β,TGF-β)超家族,1965年首次被发现并因其具有诱导骨形成的能力而得名。目前已知的BMPs数量已达30余种。成熟的BMPs是30-38kDa的蛋白,其作用通过BMPs受体与Smad蛋白磷酸化等信号传导途径而完成。BMPs可调节细胞增殖,分化及凋亡,尤其在胚胎机体发育过程中对组织器官形态发生,在成年机体生长过程中对组织器官形态的维持起重要作用。BMP最初是作为能够在体内诱导骨和软骨的形成的因子为人们所认识的,对骨骼的胚胎发育和再生修复起重要作用。现在研究认为,它们参与调节许多种细胞的增殖、分化和凋亡的生物学过程。
骨形态发生蛋白2(BMP-2)隶属于骨形态发生蛋白家族。BMP2被认为是活性最强的唯一能单独诱导成骨的因子。BMP2作为TGF-β超家族成员之一,它的主要功能是参与调控多种基因的生理活性,除了能引起多种细胞的增值、分化和凋亡外,还参与组织的再生和修复,BMP2作为最主要的骨形成调控因子,参与正常骨代谢中的各个阶段。它能够增加造血细胞的数量,提高骨髓单个核细胞中CD34下拨的数量比例,从而加快骨髓造血系统的重建,BMP2还可能参与面神经损伤后早期应激反应级修复再生活动。
近年来的研究发现BMP-2不仅能诱导成骨,而且还可以促进或抑制肿瘤细胞增殖、侵袭和转移。目前,BMP-2对恶性肿瘤细胞的增殖和转移的作用尚存在争议,可能与BMP-2的活性梯度和浓度,以及细胞内、外抑制剂有关。但是,作为当前肿瘤研究中的一个热点,通过对BMP-2的相关研究,将帮助了解BMP与恶性肿瘤发生和转移的关系,为今后的临床治疗肿瘤提供一个新的途径。目前,关于BMP-2的成骨机制已经较明确,其及其受体与相应的TGF-β配体和受体的密切关系使其与肿瘤发生及发展的关系越来越引起关注,但是其在肿瘤中的作用机制还没有完全明确。肿瘤的发生是一个多基因参与的、多因素介导的过程,BMP-2及其通路中的因子在肿瘤发生及发展中也是与别的因子或基因协同发挥作用的,该通路或许发挥着重要的作用,随着研究的进展,可以为临床治疗提供一个新的靶点。
因此,利用CRISPR/Cas9系统开发出一种BMP2基因敲除细胞模型,对促进BMP2基因功能及信号通路的相关研究,尤其是在骨骼再生修复和肿瘤治疗的靶标的研究就用极其重要的作用。
发明内容
本发明的目的是提供一种用于将骨形态发生蛋白2(BMP-2)基因敲除的CRISPR/Cas9系统,及其质粒载体,以及所得到的细胞模型。本发明应用CRISPR/Cas9技术敲除兔骨髓间充质干细胞(BMSCs)BMP2基因,采用脂质体转染方法,实现了建立BMP2基因敲除的细胞模型。骨髓间充质干细胞(BMSCs)是目前骨组织工程中应用最广泛的种子细胞,具有取材方便、对机体损伤少、与生物支架材料的粘附性能好、在体外培养具有较强的增值传代能力等优点。
本发明公开的CRISPR-Cas9靶向敲除兔BMP2基因的细胞模型,所述CRISPR/Cas9用于特异性靶向人TCAB1基因的gRNA在兔BMP2基因上的靶序列符合5’-N(20)-NGG3’或者5’-CCN-N(20)-3’的序列排列规则,兔BMP2基因上的靶序列是唯一的,其特征在于:所述gRNA在兔BMP2基因的靶向位点位于兔BMP2基因的外显子上。
上述CRISPR-Cas9靶向敲除兔BMP2基因的细胞模型,其特征在于:所述靶向位点如序列表SEQ ID NO.2序列所示。
上述CRISPR-Cas9靶向敲除兔BMP2基因的细胞模型,其特征在于:该细胞模型在制备抑制肿瘤的试剂中的应用。
上述CRISPR-Cas9靶向敲除兔BMP2基因的细胞模型,其特征在于:该细胞模型在制备促进骨骼生长的试剂中的应用。
本发明建立的BMP2基因敲除细胞模型,对于研究BMP2基因在成骨机制已经较明确,其及其受体与相应的TGF-β配体和受体的密切关系使其与肿瘤发生及发展的关系提供了有力的工具,该细胞模型能极大地促进BMP2基因功能及信号通路的相关研究,尤其是在骨骼再生修复和肿瘤治疗的靶标的研究就用极其重要的作用。该细胞模型制备方法简单,成本低廉,具有良好的稳定性和可靠性。
附图说明
附图1为T7Endonuclease I酶切结果(其中1-3泳道为不同靶向位点PCR效果;4-6泳道为不同靶向位点酶切效果);
附图2为测序结果图。
具体实施方式
下面将结合附图,对本发明的优选实施例进行详细的描述。实施例中未注明具体条件的实验方法,通常按照常规条件,例如分子克隆实验指南(第三版,J.萨姆布鲁克等著)中所述的条件,或按照制造厂商所建议的条件。
实施例1靶向兔BMP2基因的gRNA合成及载体构建
1、靶向兔BMP2基因的gRNA的选择和设计
在Genebank中找到兔BMP2基因的序列,在兔BMP2基因的第1外显子区域设计潜在靶位点。通过在线设计工具(http://crispr.mit.edu/)及gRNA的设计原则,评估兔BMP2基因序列上得分较高的靶位点设计gRNA,靶位点序列为SEQ ID NO.1-SEQ ID NO.3,并设计对应的寡核苷酸。
2、靶向兔BMP2基因的gRNA寡核苷酸序列的合成和真核表达载体的构建
将pSpCas9(BB)-2A-GFP(PX458)质粒(AddgeneplasmidID:48138,以下简称pSpCas9(BB)),用BbSI酶切,37℃水浴1小时后,1%的琼脂糖电泳,回收酶切产物(TAKARA胶回收试剂盒)。
酶切体系如下:
将靶位点序列为SEQ ID NO.1对应的两寡核苷酸退火,形成带有粘性末端的短双链DNA,反应体系如下:
将上述反应体系在200ulPCR管中混合均匀,然后将PCR管在37℃水浴锅中处理30min,再放入500ml沸水中,自然冷却至室温。
连接体系:
将带有粘性末端的双链短DNA产物连入酶切后的pSpCas9(BB)线性片段,将连接产物转化大肠杆菌DH5α感受态细胞(TakaraCode:D9057A),并涂布于Ampicillin浓度为100μg/mL的LB固体平板上培养过夜,挑取生长良好的单克隆,于15mLAmpicillin浓度为100μg/mL的LB液体培养基中,37℃振荡培养过夜,提取质粒,命名为px458-BMP2-T1。
3、无内毒素质粒DNA的制备
A、取px458-BMP2-T1质粒1μL加入100μLDH5α感受态细胞中吹匀,冰中静置20min,再放入42℃水浴90s,迅速置于冰浴中3min,加入500μLLB液体培养基,放置摇床180rpm 37℃1h,取菌液100μL均匀涂布于Ampicillin浓度为100μg/mL的LB固体培养基37℃培养过夜。
B、取单菌落于3mLAmpicillin浓度为100μg/mL的LB液体培养基中,250rpm、37℃振荡培养8小时;从中取300μL菌液接种于300mLAmpicillin浓度为100μg/mL的LB液体培养基中,并于250rpm、37℃振荡培养12~16小时;
C、收集菌液,然后在4℃、4000rpm条件下离心15min,弃上清,收集菌体,然后按照QIAGEN EndoFree Plasmid MaxiKit试剂盒说明书操作步骤提取质粒,得无内毒素的px458-BMP2质粒。
实施例2脂转染兔骨髓间充质干细胞建立BMP2基因敲除细胞模型
转染前3天,复苏兔骨髓间充质干细胞(BMSCs,购自赛业生物科技有限公司),将细胞放入加有兔骨髓间质干细胞完全培养基培养瓶中,于37℃、5%CO2的培养箱中培养,转染前一天,传代培养复苏细胞。
将培养BMSCs细胞T75瓶中的培养基吸净,加入2mL 4℃冰箱取出的0.25%胰酶,使其均匀覆盖瓶底,置于37℃培养箱中3-5min,取出,摇晃可发现细胞于底部脱离,将其全部晃下,加入3mL37℃水浴中预热的兔骨髓间质干细胞完全培养基,用10mL移液管进行吹打,吹打6-8次,不留死角,瓶口处较难吹打可将移液管对准培口,小力将培养基打出即可覆盖到接近瓶口的细胞。之后,将所有细胞吸出,置于15mL离心管中,取50ul混匀后的细胞于1.5m Leppendorf管中,加入450ul兔骨髓间质干细胞完全培养基,即为10倍稀释,混匀,取10ul细胞于计数板中计数。传代当天记为第一天,若第二天进行转染,铺900-1000万/T75;若第三天转染,铺350-400万/T75。每瓶T75加兔骨髓间质干细胞完全培养基。转染当天观察细胞密度,80-90%满即可进行转染。
采用脂质体转染法将px458-BMP2-T1转染BMSCs细胞。转染体系及试剂使用LipofectamineTM2000(invitrogen公司),转染详细步骤参照转染说明书。
转染48小时后,利用胰酶消化转染后贴壁的细胞,离心收集细胞,吸掉废液加入1ml PBS重悬细胞,取500ul放入原瓶中继续培养,剩余细胞放入1.5ml离心管,提取DNA(按照DNA提取试剂盒说明书进行)。
以提取的DNA为模板(未转染细胞DNA为对照组),扩增靶点序列。PCR反应体系如下:
PCR扩增程序:95℃预变性3min;95℃变性30s,58℃退火30s,72℃延伸40s,30个循环后72℃延伸5min,最后4℃保温。
PCR产物用T7 EndonucleaseI 50℃水浴酶切1h,酶切体系如下:
T7 Endonuclease I酶切结果如图1所示,结果显示靶位点序列为SEQ ID NO.2的靶点序列发生突变,具有高活性。
实施例3 PCR产物克隆送样测序检测
按照实施例2的方法将具有高活性的SEQ ID NO.2靶点构建载体转染的细胞稀释培养,获得细胞克隆。并鉴定细胞克隆。
提取细胞DNA并进行靶位点PCR,PCR产物用TAKARA试剂盒进行纯化后连接至PMD18-T载体上,连接体系为:
16℃下连接2小时。取感受态细胞DH5α,放置冰中融化5min,加入10ul连接产物吹匀,放置冰中20min。42℃热击90s,迅速转入冰浴中静置3min,加入500ul的LB液体培养基,置于摇床中,37℃180rpm 1h。取菌液100ul均匀涂布于LB固体培养基(含1/1000AMP),37℃培养过夜。
挑3个取单菌落,分别放入3ml LB液体培养基(含3ulAMP),37℃200rpm 12h。以1ul菌液为模板进行PCR鉴定,均为阳性。将菌液送样到上海生工进行测序,其测序结果如图2所示。结果显示靶位点序列为SEQ ID NO.2的gRNA能使目标靶位点发生移码突变,能建立稳定可靠的BMP2基因敲除细胞模型。
Claims (3)
1.CRISPR-Cas9靶向敲除兔BMP2基因的细胞模型,所述CRISPR-Cas9用于特异性靶向人TCAB1基因的gRNA在兔BMP2基因上的靶序列符合5’-N(20)-NGG3’或者5’-CCN-N(20)-3’的序列排列规则,兔BMP2基因上的靶序列是唯一的,其特征在于:所述gRNA在兔BMP2基因的靶向位点位于兔BMP2基因的外显子上。
2.根据权利要求1所述的CRISPR-Cas9靶向敲除兔BMP2基因的细胞模型,其特征在于:所述靶向位点如序列表SEQ ID NO.2序列所示。
3.根据权利要求1-2所述的CRISPR-Cas9靶向敲除兔BMP2基因的细胞模型,其特征在于:该细胞模型在制备促进骨骼生长的试剂中的应用。
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