CN111304258A - Ndufs2基因条件性点突变小鼠模型及其构建方法和应用 - Google Patents
Ndufs2基因条件性点突变小鼠模型及其构建方法和应用 Download PDFInfo
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Abstract
本发明提供了一种Ndufs2基因条件性点突变小鼠模型及其构建方法和应用,所述小鼠模型Ndufs2基因的第425位的赖氨酸突变为精氨酸。本发明构建的小鼠模型为制备预防或者治疗心脑血管相关疾病药物提供有效的实验动物模型,具有良好的医学临床应用前景,在制备预防/治疗心脑血管疾病药物中有很大的应用价值。
Description
技术领域
本发明属于基因工程技术领域,尤其是涉及一种Ndufs2基因条件性点突变小鼠模型及其构建方法和应用。
背景技术
基因突变小鼠模型的建立和应用对生命科学、基础医学的发展具有里程碑式的意义,对临床疾病的认识和治疗也起到了极大的推动作用。线粒体呼吸链功能的异常会直接或间接地引起多种严重的生理缺陷,而要想攻克这些医学难题,利用基因突变小鼠模型阐明呼吸链的结构信息和功能机理至关重要。在呼吸链CI~CIV四个复合物中,复合物CI的致病突变位点最多也最复杂,迄今为止已发现CI的100多个突变位点与疾病相关。非胰岛素依赖性糖尿病、MELAS综合征、Leigh氏脑病(亚急性坏死性脑病)以及多种癌症的发生都与CI中的突变有关。复合体I由45种蛋白质组成,有两个膜臂,Ndufs2是复合体I的一个亚基,位于复合体I亲水性膜臂,接近线粒体基底部,属亲水性蛋白质。NADH脱氢酶的亚基构成了复合体I催化核心,Ndufs2便是其催化核心之一。
关于能量代谢的科学研究是生物学领域有趣且意义重大的课题。经过一个多世纪的努力,人类对线粒体呼吸链的研究已经取得了可喜的成就。科学家已经认识到“亚基—呼吸复合物—超级复合物”这一复杂而有序的呼吸链组织形式,对呼吸体中电子传递和能量转换的分子机制有了较为深入的了解,但是,尚不清楚呼吸体亚基Ndufs2基因的点突变对超级复合物的结合与分离的影响,对适应不同的细胞状态究竟有怎样具体的生物学意义。
发明内容
有鉴于此,本发明旨在提出一种Ndufs2基因条件性点突变小鼠模型及其构建方法和应用,为制备预防或者治疗心脑血管相关疾病药物提供了新的策略。
为达到上述目的,本发明的技术方案是这样实现的:
一种Ndufs2基因条件性点突变小鼠模型,所述小鼠模型Ndufs2基因的第425位的赖氨酸突变为精氨酸。
上述Ndufs2基因条件性点突变小鼠模型的构建方法,包括如下步骤:
S1:体外转录sgRNA;
S2:构建Cas9打靶载体和donor载体;
S3:将Cas9打靶载体、sgRNA及donor载体显微注射到C57BL/6J小鼠的受精卵进行同源重组,获得F0代小鼠;
S4:经PCR和测序验证阳性的F0代小鼠与C57BL/6J小鼠交配获得可稳定遗传的F1代小鼠模型。
进一步的,所述步骤S1包括如下步骤:确定小鼠待敲除基因的靶位点,设计针对Ndufs2-T1、Ndufs2-T2的sgRNA,所述Ndufs2-T1的sgRNA序列如SEQ ID NO.1所示;所述Ndufs2-T2的sgRNA序列如SEQ ID NO.2所示。
进一步的,所述步骤S2包括如下步骤:
a、人工合成5′端带有不同酶切位点识别序列的靶序列寡核苷酸引物,通过PCR对两对引物直接退火,合成出携带不同的黏性末端的靶序列DNA短片段,将其***到CRISPR/Cas9表达载体pX330-U6-Chimeric_BB-CBh-h SpCas9 U6启动子下游,构建小鼠Ndufs2两个靶点的CRISPR/Cas9打靶载体pX330-U6-Ndufs2T1-Chimeric_BB-CBh-hSpCas9和pX330-U6-Ndufs2T2-Chimeric_BB-CBh-hSpCas9;
b、构建Donor载体,所述Donor载体的序列如SEQ ID NO.3所示。
上述Ndufs2基因条件性点突变小鼠模型在制备预防或者治疗心脑血管相关疾病药物的应用。
相对于现有技术,本发明所述的Ndufs2基因条件性点突变小鼠模型及其构建方法和应用具有以下优势:
本发明首次提供了Ndufs2基因条件性点突变小鼠模型及其构建方法和方法,未见任何国内外文献报道。本发明方法构建的小鼠模型为制备预防或者治疗心脑血管相关疾病药物提供有效的实验动物模型,具有良好的医学临床应用前景,在制备预防/治疗心脑血管疾病药物中有很大的应用价值,有较大的社会效益。
附图说明
图1为CRISPR/Cas9技术对Ndufs2基因进行基因编辑原理示意图;
图2为Ndufs2打靶位点示意图;
图3为同源重组阳性F0代小鼠PCR鉴定电泳图;
图4为F0代小鼠基因突变前测序结果;
图5为F0代小鼠基因突变后测序结果;
图6为F0代小鼠测序结果在PubMed中blast结果;
图7为心肌线粒体电子显微镜检测结果;
图8为心肌线粒体ATP检测结果;
图9为心肌线粒体复合体1检测结果;
图10为心肌线粒体Ndufs2蛋白与SUMO1蛋白结合情况检测结果。
具体实施方式
除有定义外,以下实施例中所用的技术术语具有与本发明所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
下面结合实施例及附图来详细说明本发明。
一种Ndufs2基因条件性点突变小鼠模型构建方法,原理如图1所示,具体地包括如下步骤:
(1)Ndufs2基因CRISPR/Cas9打靶位点的选择
根据小鼠的Ndufs2基因序列选择靶点,具体地,靶点选择在exon10和exon11,用于Ndufs2的敲除研究,靶点序列信息和在染色***置见表1和图2。
表1 sgRNA靶点序列
注:小写字母表示PAM序列
(2)CRISPR/Cas9表达载体的构建
CRISPR/Cas9表达载体构建方法是先人工合成5′端带有不同酶切位点识别序列的靶序列寡核苷酸引物,通过PCR对两对引物直接退火,合成出携带不同的黏性末端的靶序列DNA短片段,将其***到CRISPR/Cas9表达载体pX330-U6-Chimeric_BB-CBh-hSpCas9U6启动子下游,构建小鼠Ndufs2两个靶点的CRISPR/Cas9打靶载体pX330-U6-Ndufs2T1-Chimeric_BB-CBh-hSpCas9和pX330-U6-Ndufs2T2-Chimeric_BB-CBh-hSpCas9。
具体步骤为:
第一步,引物退火。寡聚核苷酸Ndufs2T1F、Ndufs2T1R(请给出引物的序列)直接退火,两条引物形成带有不同黏性末端的短片段,退火反应程序为90℃10min,70℃10min,自然冷却至室温,退火反应的体系如表2。
表2 引物直接退火反应体系
第二步,载体酶切。用XbaⅠ内切酶对CRISPR/Cas9骨架载体pX330-U6-Chimeric_BB-CBh-hSpCas9进行切割,程序为37℃酶切1h,65℃酶失活20min,酶切体系见表3。酶切产物用1%的琼脂胶检测,按照琼脂糖凝胶回收试剂盒说明书回收线性化的载体。
表3 酶切反应体系
第三步,链接反应。使用T4连接酶对线性化的pX330-U6-Chimeric_BB-CBh-hSpCas9和退火合成的Ndufs2T1短片段连接,16℃连接12~16h,连接反应体系见表4。
表4 连接反应体系
第四步,连接产物转化,按照常规方法进行。
第五步,测序鉴定。随机选取2-3个单克隆菌落进行扩大培养,提取质粒,用U6引物进行测序鉴定,确保***表达载体的DNA序列与设计的一致,最终判定成功获得Ndufs2打靶载体pX330-U6-Ndufs2T1-Chimeric_BB-CBh-hSpCas9和pX330-U6-Ndufs2T2-Chimeric_BB-CBh-hSpCas9。
(3)Donor质粒的构建
在不改变C57小鼠其他基因碱基序列的情况下,参考上述基因信息以及sgRNA活性靶点,设计了Donor质粒,其序列SEQ ID NO.3所示。该Do nor质粒序列由北京唯尚立德公司进行全基因合成,共计1092bp。
(4)显微注射
6~8周龄C57BL/6J雄性小鼠和雌性小鼠交配后,获取受精卵,将C as9打靶载体及donor显微注射到受精卵中,另取5只同期输卵管结扎假孕母鼠作为受体,将注射后的受精卵转移植入假孕的母鼠输卵管中。
(5)F0代小鼠鉴定
经过显微注射和胚胎移植后,F0代小鼠出生。经过PCR电泳图(图3)、测序鉴定(图4和5)以及测序结果在PubMed中的比对(图6),确定了雄鼠2号和雄鼠3号的基因型正确。
将通过上述构建方法得到的Ndufs2K425R基因鼠纯合子分别进行如下测试。
测试例1
将Ndufs2K425R基因鼠纯合子与心肌特异性Cre鼠(Myh6-Cre)交配2代后,获得Ndufs2fl/fl Myh6+/-小鼠(Mutation)与对照组Ndufs2fl/fl Myh6-/-小鼠(WT),8周鼠龄时,经腹腔注射他莫昔芬诱导(20mg/kg)5天,分离心肌线粒体,电镜检测线粒体结果如图7所示:Ndufs2基因K425R点突变小鼠,心肌线粒体肿胀,线粒体脊断裂。
测试例2
将Ndufs2K425R基因鼠纯合子与心肌特异性Cre鼠(Myh6-Cre)交配2代后,获得Ndufs2fl/fl Myh6+/-小鼠与对照组Ndufs2fl/fl Myh6-/-小鼠,8周鼠龄时,经腹腔注射他莫昔芬诱导(20mg/kg)5天,分离心肌线粒体,ATP活性检测结果如图8所示:Ndufs2基因K425R点突变小鼠,ATP活性明显下降。
测试例3
将Ndufs2K425R基因鼠纯合子与心肌特异性Cre鼠(Myh6-Cre)交配2代后,获得Ndufs2fl/fl Myh6+/-小鼠与对照组Ndufs2fl/fl Myh6-/-小鼠,8周鼠龄时,经腹腔注射他莫昔芬诱导(20mg/kg)5天,分离心肌线粒体,线粒体复合体Ⅰ活性检测结果如图9所示:Ndufs2基因K425R点突变小鼠,线粒体复合体Ⅰ活性明显下降。
测试例4
将Ndufs2K425R基因鼠纯合子与心肌特异性Cre鼠(Myh6-Cre)交配2代后,获得Ndufs2fl/fl Myh6+/-小鼠与对照组Ndufs2fl/fl Myh6-/-小鼠,8周鼠龄时,经腹腔注射他莫昔芬诱导(20mg/kg)5天,分离心肌线粒体,免疫共沉淀检测结果如图10所示:Ndufs2基因K425R点突变小鼠,Ndufs2蛋白与SUMO1蛋白的结合能力明显下降。
从测试例1-4的测试结果可以得出,验证基因Ndufs2在县脑血管疾病中起着重要的作用。因此,Ndufs2基因K425R点突变小鼠模型可作为用于研究心脑血管相关疾病发生及发展恶化的模型,并为相关药物的制备提供了便利。
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,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (5)
1.Ndufs2基因条件性点突变小鼠模型,其特征在于:所述小鼠模型Ndufs2基因的第425位的赖氨酸突变为精氨酸。
2.权利要求1所述的Ndufs2基因条件性点突变小鼠模型的构建方法,其特征在于:包括如下步骤:
S1:体外转录sgRNA;
S2:构建Cas9打靶载体和donor载体;
S3:将Cas9打靶载体、sgRNA及donor载体显微注射到C57BL/6J小鼠的受精卵进行同源重组,获得F0代小鼠;
S4:经PCR和测序验证阳性的F0代小鼠与C57BL/6J小鼠交配获得可稳定遗传的F1代小鼠模型。
3.根据权利要求2所述的Ndufs2基因条件性点突变小鼠模型的构建方法,其特征在于:所述步骤S1包括如下步骤:确定小鼠待敲除基因的靶位点,设计针对Ndufs2-T1、Ndufs2-T2的sgRNA,所述Ndufs2-T1的sgRNA序列如SEQ ID NO.1所示;所述Ndufs2-T2的sgRNA序列如SEQ ID NO.2所示。
4.根据权利要求3所述的Ndufs2基因条件性点突变小鼠模型的构建方法,其特征在于:所述步骤S2包括如下步骤:
a、人工合成5′端带有不同酶切位点识别序列的靶序列寡核苷酸引物,通过PCR对两对引物直接退火,合成出携带不同的黏性末端的靶序列DNA短片段,将其***到CRISPR/Cas9表达载体pX330-U6-Chimeric_BB-CBh-h SpCas9 U6启动子下游,构建小鼠Ndufs2两个靶点的CRISPR/Cas9打靶载体pX330-U6-Ndufs2T1-Chimeric_BB-CBh-hSpCas9和pX330-U6-Ndufs2T2-Chimeric_BB-CBh-hSpCas9;
b、构建Donor载体,所述Donor载体的序列如SEQ ID NO.3所示。
5.一种权利要求1所述的Ndufs2基因条件性点突变小鼠模型在制备预防或者治疗心脑血管相关疾病药物的应用。
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