CN106399369B - 构建在海马体区域特异性敲除IKKα基因的小鼠模型的方法及打靶载体和试剂盒 - Google Patents
构建在海马体区域特异性敲除IKKα基因的小鼠模型的方法及打靶载体和试剂盒 Download PDFInfo
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Abstract
本发明公开了构建在海马体区域特异性敲除IKKα基因的小鼠模型的方法及打靶载体和试剂盒。本发明利用Cre‑LoxP重组系统构建在特定小鼠组织中敲除IKKα基因,从而建立IKKα基因敲除的转基因小鼠,以便在动物的整体水平研究NF‑κB信号通路与疾病尤其是肿瘤的作用机制。同时,为便于确定和监测IKKα基因在海马体区域的特异性敲除是否会对小鼠行为学造成影响,在本发明中利用红色荧光素酶RFP720连接至转基因载体中。即,本发明通过基因打靶技术,基于iRFP720近红外荧光蛋白和Cre‑LoxP基因敲除系统降低小鼠海马体区域内源性IKKα基因的表达,进而通过动物成像系统监测转基因小鼠的活体成像。
Description
技术领域
本发明属于影像学、病理学、生理学,遗传学,分子生物学以及行为学技术领域。具体涉及构建在海马体区域特异性敲除IKKα基因的小鼠模型的方法及打靶载体和试剂盒。
背景技术
IKKα是组成IKK复合物的调节亚基,IKK复合物能够影响NF-κB信号通路的活化。在胶质瘤(一种发生在脑部的肿瘤)研究中表明,NF-κB等与炎症相关的信号通路处于异常活化状态,目前关于IKKα在脑部肿瘤中的机制研究并不清楚,其敲除后是否会影响脑部肿瘤的发生发展尚不明确,同时其作为炎症相关分子,在正常哺乳动物海马体区域完全敲除后是否会影响其行为学功能并不清楚。因此,利用动物模型研究脑部IKKα异常活化而引发的疾病,可以为人类脑部肿瘤及可能伴随的异常行为学功能的诊断,治疗提供理论基础。
IKK复合物主要有三种亚基组成,分子量大小为700-900KD,三种亚基分别为IKKα(85KD),IKKβ(87KD),IKKγ(48KD),其中IKKα和IKKβ为催化亚基,IKKγ为结构亚基。IKKα和IKKβ的基因结构相似,但具有不同的生物学活性。IKKα蛋白可与TNFα受体家族成员中的NF-κB受体激活蛋白作用,进而影响NF-κB蛋白与DNA的结合活性。而IKKβ蛋白主要与炎症相关。IKK复合物的活化主要与能引起IKK复合物磷酸化修饰的生物学进程相关。在肿瘤的发生发展过程中,NF-κB的抗凋亡,促增殖和免疫激活功能是致使正常细胞癌变的重要因素。在胶质瘤细胞中,NF-κB处于异常活化和持续激活状态。正常细胞中,NF-κB参与的炎症反应对于维持机体正常防御外界因素感染以及修复机制有重要作用。完全敲除IKKα基因,会导致小鼠胚胎致死。
Cre-LoxP系统是由一个单一的Cre重组酶和被称为LoxP序列的靶向基因敲除序列组成。Cre重组酶有70%的重组效率,不借助任何辅助因子,可作用于多种结构的DNA底物,如线形、环状甚至超螺旋DNA。它是一种位点特异性重组酶,能介导两个LoxP位点(序列)之间的特异性重组,使LoxP位点间的基因序列被删除或重组。Cre-Lox重组系统在目前存在的转基因动物模型的表型和基因型之间的联系实现最佳的实验控制Cre重组酶可以识别LoxP序列,通过DNA重组的原理,可以将两个LoxP序列之间的片断,即DNA片断去除。如果是没有Cre,LoxP只是在特定的基因片段两侧,不影响基因片段的活性。一旦Cre酶有活性,就可以把LoxP中间所夹的序列去除,即条件性基因敲除。Cre重组酶介导两个LoxP位点间的重组是一个动态、可逆的过程,可以分成三种情况:
1、如果两个LoxP位点位于一条DNA链上,且方向相同,Cre重组酶能有效切除两个LoxP位点间的序列;
2、如果两个LoxP位点位于一条DNA链上,但方向相反,Cre重组酶能导致两个LoxP位点间的序列倒位;
3、如果两个LoxP位点分别位于两条不同的DNA链或染色体上,Cre酶能介导两条DNA链的交换或染色体易位。
目前,国内、外实验动物成像的主要手段包括结构成像(解剖成像)及功能成像(分子成像)。活体光学成像与micro-CT、MRI(magnetic resonance imaging)、ultrasound等结构成像手段结合,能为动物实验提供更客观的数据、更确切的分子生物特性。活体成像技术可以从影响基因表达的各个不同的层面进行基因研究,可应用于某种疾病的基因表达、转基因动物实验以及基因治疗等,可将靶基因进行荧光素酶标记后转入动物体内,形成研究所需的各种小动物疾病模型,包括肿瘤,免疫系统疾病,感染性疾病等。近红外光的最大吸收波长和发射波长为650-900nm,活体成像时,哺乳动物组织因其透光性和深度组织的背景干扰,普通的荧光标签不能很好的反应其生理功能,近红外荧光是近年来在影像学研究中较为热门的研究之一,因其可减少自发荧光和低光散射使其可作为穿透深度组织的优选标签。iRFP720是一种近红外荧光蛋白基因,从细菌的感光细胞中分离得到,可用于基因工程编辑构建打靶载体的标签,目前对于iRFP720作为一种近红外红色荧光标签构建动物模型的研究较为缺乏。
发明内容
本发明旨在克服现有技术的不足,提供构建在海马体区域特异性敲除IKKα基因的小鼠模型的方法及打靶载体和试剂盒。
为了达到上述目的,本发明提供的技术方案为:
所述构建在海马体区域特异性敲除IKKα基因的小鼠模型的方法包括如下步骤:
(1)构建打靶载体pBR322-MCS-2S-Chuk-KI,所述打靶载体pBR322-MCS-2S-Chuk-KI的序列如SEQ ID NO.1所示;
(2)将iRFP720红色荧光蛋白基因连接至打靶载体,然后导入小鼠,构建携带有iRFP720基因的IKKα基因knock in杂合型小鼠;
(3)将IKKα基因knock in杂合型转基因小鼠与CamKIIalpha-cre T29-1转基因小鼠分别与C57野生型小鼠扩大繁殖,然后将IKKα基因knock in杂合型转基因小鼠与CamKIIalpha-cre T29-1转基因小鼠交配获得杂合IKKαF/+·Camk2α.Cre-LoxP小鼠,再利用杂合型小鼠自交获得纯合型IKKαF/F·Camk2α.Cre-LoxP小鼠;通过小动物活体成像仪分别观察杂合型及纯合型Cre-LoxP小鼠的红色荧光,选择可以检测到iRFP720红色荧光蛋白表达的小鼠,即为在海马体区域特异性敲除IKKα基因的小鼠模型。
构建打靶载体pBR322-MCS-2S-Chuk-KI的试剂盒中包括如下引物:
Chuk-A-F:ACGCGTCGACGAACACTTTTCTAAGGCTG(SEQ ID NO.2);
Chuk A-R:CGGGGTACCCACTTCCTTTTGAAATTAG(SEQ ID NO.3);
Chuk-B-F:CGGGGTACCTATTTTAGACCGTCAGTC(SEQ ID NO.4);
Chuk-B-R:CGCGGATCCTTGGCCAAGCCATGGCG(SEQ ID NO.5);
RFP720-F:AATATGGCCACACGGTCGCCACCATGGCG(SEQ ID NO.6);
RFP720-R:AGGGAATCTACACAGTCGCGGCCGCTCACTC(SEQ ID NO.7);
En2SA-F:GACTGTGGCCATATTATCATCG(SEQ ID NO.8);
BGH-R:CTGTGTAGATTCCCTGGGACC(SEQ ID NO.9)。
下面对本发明作进一步说明:
本发明的详细步骤如下:
(一)打靶载体的构建
从Ensembl数据库中(http://www.ensembl.org/index.html)获得Chuk基因组序列,根据基因组序列设计打靶载体。小鼠Chuk-001转录本共有21个外显子,根据生物信息学分析,knockin元件、neo、frt等元件插入第1~2内含子。运用ET克隆的方法构建Chuk基因1~2内含子插入knockin元件的打靶载体。大致过程如下:以BAC质粒为模板DNA,PCR扩增A、B、C、D四个小同源臂。A臂和B臂克隆至pBR322-2S的SalI、BamHI的位点,该质粒经KpnI单酶切后,获得retrieve质粒;通过同源重组方式,经氨苄青霉素筛选,获得克隆了BAC的A区域到B区域之间DNA序列的质粒,该序列包含5’和3’同源臂;通过PCR和酶切克隆,将knockin序列lox2272-loxp-TRE-(pA-iRFP670-IRES-En2SA-lox2272-loxp)(Rv)构建完成,将Chuk的C臂和knockin元件克隆至PL451的KpnI、EcoRI的位点,将Chuk的D臂克隆至PL451的BamHI、NotI的位点,该重组质粒经KpnI/NotI双酶切后,分离获得含C-KI-Neo-D片断,然后经过同源重组knock in到上述获得的含Retrieve质粒中,构建载体pBR322-MCS-2S-Chuk-KI,并用限制性内切酶及测序鉴定。酶切结果如图1所示。
(二)建立IKKα基因konck in小鼠模型
本部分实验性动物小鼠购自上海南方生物科技发展有限公司,根据本研究团队要求公司将iRFP720红色荧光蛋白连接至打靶载体,该载体只有在与Cre-LoxP建立稳定系统后,iRFP720红色荧光蛋白会发生反转录,红色荧光蛋白才会得以表达,在动物成像系统中看到红色荧光,公司提供携带有iRFP720基因的IKKα基因knock in杂合型小鼠。打靶载体结构图和序列如图2所示。
(三)嵌合体小鼠育种及PCR鉴定cre-LoxP小鼠模型
我们将IKKα基因knock in杂合型转基因小鼠与CamKIIalpha-cre T29-1(CamK2a.Cre)转基因小鼠分别与C57野生型小鼠扩大繁殖,然后将IKKα基因knock in杂合型转基因小鼠与CamKIIalpha-cre T29-1转基因小鼠交配获得杂合IKKαF/+·Camk2α.Cre小鼠,再利用杂合型小鼠自交获得纯合型IKKαF/F·Camk2α.Cre小鼠,通过小动物活体成像仪分别观察杂合型及纯合型Cre-LoxP小鼠的红色荧光,发现杂合型与纯合型小鼠,当Cre重组酶在杂合型或纯合型小鼠整合至染色体上时,都可以检测到红色荧光iRFP720的表达。PCR引物鉴定序列如表1所示。
表1
Cre-Loxp系统杂交策略
a.IKKαknock in杂合型转基因小鼠育种策略
b.CamKIIalpha-cre小鼠育种策略
c.IKKαF/F·Camk2α.Cre系统构建策略
(四)动物成像仪检测小动物活体成像荧光。
iRFP720最大激发光波长为702nm,发射光波长为720nm。因此,我们选定iRFP720作为打靶基因载体的标签,构建动物模型,以便在后续动物成像中得以做到实时监测小动物生理学变化,iRFP720可穿透皮肤和骨骼组织,在活体成像仪的近红外区检测到红色荧光。
CamKIIalpha-cre T29-1转基因小鼠携带有鼠钙/钙调蛋白激酶IIalpha(Camk2α)启动子序列,可在小鼠前脑,特别是在海马体CA1椎体细胞层表达Cre重组酶活性。当与携带有LoxP位点序列的小鼠杂交后,Cre介导的重组发生在子代海马体椎体细胞层。CaMKIIalpha-cre T29-1纯合转基因小鼠,体型肥硕,大小正常,无明显身体异常或行为异常。该转基因小鼠载体含有8.5kbp序列,包括编码鼠钙/钙调蛋白激酶IIalpha(Camk2α)启动子序列,Cre重组酶序列,SV40多聚腺苷酸化位点序列,构建转基因小鼠时将载体显微注射至BCF1的卵细胞中,与C57BL/6小鼠杂交,并回交至10代以上获得转基因小鼠。
本发明通过动物活体成像监测小鼠海马体区域特异性敲除IKKα基因后,对小动物活体成像的近红外光进行实时监测并研究其对脑部肿瘤发生发展的影响,所涉及解决的技术问题主要有打靶载体的构建,嵌合体小鼠育种及PCR鉴定小鼠基因型,Cre-LoxP系统的建立和动物活体成像观察纯合Cre-LoxP小鼠iRFP720红色荧光信号。提供了一种可以从近红外动物成像仪中监测小鼠海马体区域特异性敲除IKKα基因的可视化小鼠模型。
本发明利用Cre-LoxP重组系统构建在特定小鼠组织中敲除IKKα基因,从而建立IKKα基因敲除的的转基因小鼠,以便在动物的整体水平研究NF-κB信号通路与疾病尤其是肿瘤的作用机制。同时,为便于确定和监测IKKα基因在海马体区域的特异性敲除是否会对小鼠行为学造成影响,在本发明中利用红色荧光素酶RFP720连接至转基因载体中。即,本发明通过基因打靶技术,基于iRFP720近红外荧光蛋白和Cre-LoxP基因敲除系统降低小鼠海马体区域内源性IKKα基因的表达,进而通过动物成像系统监测转基因小鼠的活体成像。
附图说明
图1:打靶载体用EcoRV酶酶切后的电泳图;M:1kb DNA ladder;1:1号质粒;2:2号质粒;
图2:靶载体图谱;
图3:IKKαknock in转基因小鼠PCR鉴定基因型结果图
图4:CamK2α小鼠PCR鉴定基因型结果图
图5:IKKαF/F·Camk2α.Cre小鼠PCR鉴定基因型结果图
具体实施方式
(一)转基因小鼠IKKα和CamK2α的PCR鉴定
小鼠出生三周后,置于1.5ml EP管中,加500μl裂解液和20μl蛋白酶K,放入1.5mlEP管中,60℃条件下,在恒温金属浴中过夜孵育,次日500g离心吸取上清液,测量得到的粗提gDNA浓度,并将gDNA浓度稀释至30ng/μl,以便后续PCR反应验证。按照上述引物进行PCR验证,PCR产物为643bp表明IKKαknock in已经整合至小鼠的染色体基因组中,纯合型只有单纯643bp条带,杂合型则有299bp和643bp两条带型,野生型小鼠则只有299bp,为阴性小鼠。PCR产物若检测到<100bp条带,表明CamK2α已经整合至小鼠染色体基因组中,该小鼠为CamK2α转基因小鼠,通过普通PCR鉴定不能区分CamK2α小鼠的纯合型与杂合型,但只要可检测出<100bp的条带,则可视为CamK2α转基因小鼠。随后再将新生的子代与野生型小鼠交配2代,获得稳定转基因小鼠。结果见图3和图4。
(二)Cre-LoxP系统的构建
在获得稳定转基因型小鼠IKKα和CamK2α后,将转基因小鼠IKKα和CamK2α合笼,以便获得子代为杂合型IKKαF/+·Camk2α.Cre小鼠,抽提鼠尾gDNA通过普通PCR方法鉴定基因型。按照上述引物进行PCR验证,PCR产物为643bp和<100bp,并能检测出299bp或324bp的小鼠即为阳性杂合型IKKαF/+·Camk2α.Cre小鼠。出生的阳性小鼠通过与阳性之间进行自交,即杂合型与杂合型小鼠配种,获得纯合型IKKαF/F·Camk2α.Cre小鼠,PCR鉴定若小鼠可检测出643bp,759bp和<100bp条带则为纯合型IKKαF/F·Camk2α.Cre,759bp条带表明Cre重组酶与IKKαknock in已整合至小鼠的同意染色体基因组中。结果见图5。
(三)小动物活体成像
利用小动物活体成像仪,将参数设置成激发光波长为680nm或715nm,只要携带有759bp带型即可看到红色荧光,759bp表示Cre与knock in元件在同一染色体上,可以反转iRFP720的表达,小动物成像仪可见红色荧光。
Claims (3)
1.一种构建在海马体区域特异性敲除IKKα基因的小鼠模型的方法,其特征在于,所述方法包括如下步骤:
(1)构建打靶载体pBR322-MCS-2S-Chuk-KI,所述打靶载体pBR322-MCS-2S-Chuk-KI的序列如SEQ ID NO.1所示;
(2)将iRFP720红色荧光蛋白基因连接至打靶载体,然后导入小鼠,构建携带有iRFP720基因的IKKα基因knock in杂合型小鼠;
(3)将IKKα基因knock in杂合型转基因小鼠与CamKIIalpha-cre T29-1转基因小鼠分别与C57野生型小鼠扩大繁殖,然后将IKKα基因knock in杂合型转基因小鼠与CamKIIalpha-cre T29-1转基因小鼠交配获得杂合IKKαF/+.Camk2α.Cre-LoxP小鼠,再利用杂合型小鼠自交获得纯合型IKKαF/F.Camk2α.Cre-LoxP小鼠;通过小动物活体成像仪分别观察杂合型及纯合型Cre-LoxP小鼠的红色荧光,选择可以检测到iRFP720红色荧光蛋白表达的小鼠,即为在海马体区域特异性敲除IKKα基因的小鼠模型。
2.构建权利要求1所述小鼠模型的打靶载体,其特征在于,所述打靶载体为pBR322-MCS-2S-Chuk-KI,序列如SEQ ID NO.1所示。
3.构建权利要求2所述打靶载体的试剂盒,其特征在于,所述试剂盒中包括如下引物:Chuk-A-F:ACGCGTCGACGAACACTTTTCTAAGGCTG;
Chuk A-R:CGGGGTACCCACTTCCTTTTGAAATTAG;
Chuk-B-F:CGGGGTACCTATTTTAGACCGTCAGTC;
Chuk-B-R:CGCGGATCCTTGGCCAAGCCATGGCG;
RFP720-F:AATATGGCCACACGGTCGCCACCATGGCG;
RFP720-R:AGGGAATCTACACAGTCGCGGCCGCTCACTC;
En2SA-F:GACTGTGGCCATATTATCATCG;
BGH-R:CTGTGTAGATTCCCTGGGACC。
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