CN116941578A - 一种原发性牙齿萌出障碍的pth1r基因点突变小鼠模型的构建方法及应用 - Google Patents
一种原发性牙齿萌出障碍的pth1r基因点突变小鼠模型的构建方法及应用 Download PDFInfo
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Abstract
本发明公开了一种原发性牙齿萌出障碍的PTH1R基因点突变小鼠模型的构建方法,包括以下步骤:基于目标基因PTH1Rc.1325_1336del突变位点设计gRNA;根据所述gRNA序列设计供体寡核苷酸供体DNA;将小鼠Pth1r基因的gRNA、含有c.1325_1336del突变的供体DNA和Cas9共同注射到小鼠受精卵中,再将受精卵移植到假孕母鼠子宫中,获得F0代小鼠;经PCR和测序验证正确的F0代阳性杂合子小鼠与C57BL/6JGpt小鼠交配获得可稳定遗传的阳性F1代小鼠,F1代杂交得到F2,经基因测序验证得到Pth1r基因c.1325_1336del的原发性牙齿萌出障碍小鼠模型。本发明的动物模型为研究PTH1R基因c.1325_1336del突变人群PFE的发病机制的理想载体,弥补了现有动物模型的不足,为PFE相关的发病机制及干预研究提供了良好的基础,可以为PFE的相关研究提供更加深入的见解。
Description
技术领域
本发明涉及动物模型技术领域,尤其涉及一种原发性牙齿萌出障碍的PTH1R基因点突变小鼠模型的构建方法及应用。
背景技术
原发性牙齿萌出障碍(Primary failure oferuption,PFE)是一种罕见的常染色体显性遗传病,发病率仅0.06%。PFE由Proffit在1981年正式提出,定义为“没有固连的牙齿因萌出机制异常导致牙齿部分或全部不能萌出,是造成开合的病因之一”。PFE患者没有系统性的全身疾病,也不存在干扰牙齿萌出的局部障碍因素。PFE的诊治困难,目前临床上缺乏治疗PFE的手段。PFE患牙对正畸力几乎无反应,受正畸力的PFE患牙不发生移动或仅倾斜移动1-2mm。
大量研究已经证实,PFE与甲状旁腺激素受体1基因(parathyroid hormonereceptor 1,PTH1R)突变有关。PTH1R基因位于人的第3号常染色体短臂上(3p22-21.1),在人体的肾脏、肺、口腔等多个器官中均有表达。其编码的是甲状旁腺激素受体1(PTHR1)或称PPR,一种B类G蛋白偶联受体。PTH1R基因通过调控牙骨质及牙周膜附着组织的形成影响牙根形成及牙齿萌出,是牙齿发育及萌出中的关键因素。PTH1R基因c.1325_1336del突变位点在一个PFE家系中首次被发现,父子三人均携带这一突变位点,证实其发生稳定遗传。该位点的致病性明确,但致病机制未明。采用基因工程技术构建模拟人类PTH1R基因c.1325_1336del突变位点的动物模型有望成为解决PFE相关科学问题的关键。
已有国内外研究通过建立PTH1R基因点突变的细胞模型来研究PFE的发病机制。现有细胞模型有:Cos7细胞(1092delG PTH1R)、Hela细胞(356C>T、395C>T、439C>T和1148G>A)、非洲爪蟾卵母细胞(PTH1R/Trp339stop)。这些细胞模型虽然模拟了PTH1R基因的点突变,但不足之处在于:实验所采用的肾源性细胞或Hela细胞与牙齿发育的相关性较弱,与牙源性细胞相比同质性差别较大,不能准确反映PTH1R基因点突变在牙齿发育过程中所发挥的作用,以此来研究PFE的发病机制存在明显的局限性。
现有关于PTH1R基因突变的动物模型有:通过Prx1启动子条件性缺失Pth1r构建的PFE小鼠模型;将表达PTHrP牙囊细胞中的Pth1r双等位基因失活后建立的PFE小鼠模型;选择性敲除表达Osx基因细胞中的Pth1r获得的PFE小鼠模型。这些动物模型多采用条件性敲除技术,不足之处在于:1、条件性敲除小鼠敲除的是全长PTH1R基因,而PFE患者多为PTH1R基因的某个片段异常;2、PFE患者为全身的PTH1R基因发生异常,而条件性敲除小鼠为局部组织中的Pth1r基因缺失,不能反映基因突变对其他器官造成的影响。因此,条件性敲除获得的小鼠在用于研究临床PFE患者的发病机制时存在明显缺陷。
通过上述分析,现有技术存在的问题及缺陷为:现有关于PTH1R基因突变的动物模型从分子遗传学层面不能准确模拟临床PFE患者发病的病理过程。由于PFE的发病机制尚不清楚,缺乏有效的诊治手段,亟需一种能够模拟人PFE发病的动物模型。
发明内容
本发明的目的是提出一种原发性牙齿萌出障碍的PTH1R基因点突变小鼠模型的构建方法及应用,从而更准确的模拟人类PTH1R基因突变后导致PFE的过程。
本发明的目的将通过以下技术方案得以实现:
本发明的目的之一在于提供一种原发性牙齿萌出障碍的PTH1R基因点突变小鼠模型的构建方法,包括以下步骤:
(1)基于目标基因PTH1R c.1325_1336del突变位点设计gRNA;
(2)根据所述gRNA序列设计供体寡核苷酸供体DNA;
(3)将小鼠Pth1r基因的gRNA、含有c.1325_1336del突变的供体DNA和Cas9共同注射到小鼠受精卵中,再将受精卵移植到假孕母鼠子宫中,获得F0代小鼠;
(4)经PCR和测序验证,将所述F0代小鼠划分为野生型小鼠或阳性杂合子小鼠,然后将所述阳性杂合子小鼠与C57BL/6JGpt小鼠交配获得可稳定遗传的阳性F1代小鼠;
(5)将所述F1代小鼠杂交得到F2代小鼠,通过基因鉴定可获得3种Pth1r基因型:Pth1r野生型、Pth1r突变杂合子、Pth1r突变纯合子。
进一步的,所述gRNA的序列如SEQ ID NO.1所示:
gRNA-A1:AGAGCATCTCATAGTGCATC-TGG;
所述供体DNA的序列如SEQ ID NO.2所示:
Oligo:
CCGTCTTCATGGCCTTGCCGTACACCGAGGTCTCAGGGACACT GTGGCAGATCCAGATGCTCTTCAACTCCTTCCAGGTGTGCGGCCTG CCTGGGGTTCTGGGGAGTGCACGGGGTGGGGC。
进一步的,所述gRNA、供体DNA和Cas9的混合物中,gRNA的浓度为2pmol/μl,供体DNA的浓度为100ng/μl,Cas9的浓度为30ng/μl。
进一步的,所述基因型鉴定或筛选步骤中,繁育的小鼠出生后1-2周剪尾并提取基因组DNA,通过PCR反应,将得到的PCR产物进行测序、基因型鉴定或筛选;将PCR扩增产物进行测序,得到小鼠的基因型。
进一步的,所述提取基因组DNA过程中使用TaKaRa MiniBEST通用基因组DNA提取试剂盒(Ver.5.0_Code No.9765)获得高纯度的基因组DNA。
进一步的,所述PCR反应引物为:
正向引物(F1)的核苷酸序列如SEQ ID NO.3所示:5'-AGGAGAAGCGACTGTTGTGAGG-3';
反向引物(R1)的核苷酸序列如SEQ ID NO.4所示:5'-CCATTGCAGAAAGTATATGATG-3'。
进一步的,所述PCR反应所使用的Taq DNA聚合酶是GreenTaqMix(Vazyme P131)。
本发明的目的之二在于上述的PTH1R基因点突变小鼠模型的构建方法构建得到的动物模型在制备研究原发性牙齿萌出障碍疾病的产品中的用途。
本发明的目的之三在于上述的PTH1R基因点突变小鼠模型的构建方法构建得到的动物模型在验证治疗原发性牙齿萌出障碍药物的药效中的应用。
本发明的目的之三在于上述的PTH1R基因点突变小鼠模型的构建方法构建得到的动物模型在评估牙、骨发育及其他基因富集组织器官表型分析中的应用,或者在评估颌骨骨髓间充质干细胞增殖、凋亡及成骨的影响研究中的应用。
本发明的突出效果为:
本发明基于CRISPR/Cas9获得PTH1R基因定点突变动物模型,可以准确模拟人类PTH1R基因c.1325_1336del突变位点患者的发病情况,且不会为机体及后续研究结果引入不可控影响。因此该模型为研究PTH1R基因c.1325_1336del突变人群PFE的发病机制的理想载体。此外,本发明方法构建的动物模型可控性高、重复性好、更准确的模拟了人类PTH1R基因c.1325_1336del突变导致PFE的过程,弥补了现有动物模型的不足,为PFE相关的发病机制及干预研究提供了良好的基础,可以为PFE的相关研究提供更加深入的见解。
以下便结合实施例,对本发明的具体实施方式作进一步的详述,以使本发明技术方案更易于理解、掌握。
附图说明
图1为本发明实施例中人源NM_000316.3与鼠源NM_001083936转录本的氨基酸序列同源性对比结果;
图2为本发明实施例中基因型筛选时PCR扩增产物的电泳结果图,图中MT表示突变型,WT为野生型对照,泳道11、12、19、23为F1代小鼠的PCR扩增产物电泳结果;
图3为本发明实施例中基因型筛选时PCR扩增产物测序结果图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是为了更好地进一步理解本发明,并不局限于所述最佳实施方式,不对本发明的内容和保护范围构成限制,任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品,均落在本发明的保护范围之内。
实施例中未注明具体实验步骤或条件者,按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规试剂产品。
实施例
查阅NCBI数据库可知:经同源性比对分析发现,人源NM_000316.3与鼠源NM_001083936转录本的氨基酸序列相似度为89.04%,如图1所示。鉴于此2个转录本同源性非常高,且氨基酸序列改变处于同蛋白质结构域,因此,本发明选择小鼠来构建基于CRISPR/Cas9获得PTH1R基因定点突变动物模型。
小鼠Pth1r基因(Genbank登录号:NM_001083936;Ensembl:ENSMUSG00000032492.15)位于小鼠的9号染色体上,共含14个外显子。目标基因位于第13个外显子上,外显子13被选做目标位点,设计gRNA序列如SEQ ID NO.1所示:
gRNA-A1:AGAGCATCTCATAGTGCATC-TGG。
根据所述gRNA序列设计供体寡核苷酸供体DNA,供体DNA的序列如SEQ ID NO.2所示:
Oligo:
CCGTCTTCATGGCCTTGCCGTACACCGAGGTCTCAGGGACACT GTGGCAGATCCAGATGCTCTTCAACTCCTTCCAGGTGTGCGGCCTG CCTGGGGTTCTGGGGAGTGCACGGGGTGGGGC。
将小鼠Pth1r基因的gRNA、含有c.1325_1336del突变的供体DNA和Cas9进行混合(其中gRNA的浓度为2pmol/μl,供体DNA的浓度为100ng/μl,Cas9的浓度为30ng/μl),使用显微注射仪注射到C57BL/6JGpt小鼠(赛业生物科技有限公司)单细胞受精卵胞浆中。然后将注射后存活的受精卵移植到C57BL/6JGpt假孕母鼠子宫中,出生的小鼠为被编辑的F0代小鼠,繁育的小鼠出生1周后,剪尾并收集鼠尾,抽提基因组DNA,通过PCR反应,进行基因型筛选,PCR反应体系和反应程序如下:
PCR反应体系
PCR反应程序
PCR所采用的引物为:
正向引物(F1)的核苷酸序列如SEQ ID NO.3所示:5'-AGGAGAAGCGACTGTTGTGAGG-3';
反向引物(R1)的核苷酸序列如SEQ ID NO.4所示:5'-CCATTGCAGAAAGTATATGATG-3'。
在标准条件下,在25μL体积中进行35个循环的PCR,将上面列出的两个引物添加到每个反应中。所使用的Taq DNA聚合酶是Green Taq Mix(Vazyme P131)。用于PCR基因分型的两种对照是:水对照组:未添加DNA模板。野生型对照:小鼠基因组DNA。
将上述获得的PCR扩增产物进行测序由赛业(广州)生物科技有限公司完成。
经PCR和测序验证正确的F0代阳性小鼠与C57BL/6JGpt小鼠交配获得可稳定遗传的F1代阳性小鼠模型(杂合子),F1代杂交得到F2代通过基因鉴定可获得3种pth1r基因型:pth1r野生型、pth1r突变杂合子、pth1r突变纯合子。
Pth1r基因突变小鼠扩群及基因鉴定:P14天小鼠剪尾→DNA提取→针对本项目缺失片段设计相应的PCR引物→PCR扩增→扩增DNA片段测序鉴定样品的表达水平。PCR反应步骤同上述。提取基因组DNA过程中使用TaKaRa MiniBEST通用基因组DNA提取试剂盒(Ver.5.0_CodeNo.9765)获得高纯度的基因组DNA。PCR扩增产物通过电泳,电泳结果如图2所示,图中MT表示突变型,WT为野生型对照,泳道11、12、19、23为F1代小鼠的PCR扩增产物电泳结果,收集该条带的扩增产物进行测序验证,测序结果如图3所示,F1代小鼠的泳道11、12、19、23的PCR产物具有c.1325_1336del缺失突变,通过基因型筛选后得到的阳性子代小鼠为F1代杂合子。
pth1r突变小鼠种群扩增:通过F1杂交可获得野生型、突变杂合子和突变纯合子。为获得足量的突变杂合子,可以采用杂合子×杂合子、杂合子×野生型策略获得;为获得足量的纯合子,如果纯合子胚胎或未性成熟致死,则采用杂合子×杂合子策略获得,如果纯合子可育,则通过纯合子×纯合子实现扩群。
再次重复对Pth1r基因突变小鼠进行基因鉴定,确定小鼠为缺失目标序列c.1325_1336del的PFE模型小鼠,说明本发明的PTH1R基因点突变动物模型构建成功。
以上,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (10)
1.一种原发性牙齿萌出障碍的PTH1R基因点突变小鼠模型的构建方法,其特征在于包括以下步骤:
(1)基于目标基因PTH1R c.1325_1336del突变位点设计gRNA;
(2)根据所述gRNA序列设计供体寡核苷酸供体DNA;
(3)将小鼠Pth1r基因的gRNA、含有c.1325_1336del突变的供体DNA和Cas9共同注射到小鼠受精卵中,再将受精卵移植到假孕母鼠子宫中,获得F0代小鼠;
(4)经PCR和测序验证,将所述F0代小鼠划分为野生型小鼠或阳性杂合子小鼠,然后将所述阳性杂合子小鼠与C57BL/6JGpt小鼠交配获得可稳定遗传的阳性F1代小鼠;
(5)将所述F1代小鼠杂交得到F2代小鼠,通过基因鉴定可获得3种Pth1r基因型:Pth1r野生型、Pth1r突变杂合子、Pth1r突变纯合子。
2.根据权利要求1所述的一种原发性牙齿萌出障碍的PTH1R基因点突变小鼠模型的构建方法,其特征在于:
所述gRNA的序列如SEQ ID NO.1所示:
gRNA-A1:AGAGCATCTCATAGTGCATC-TGG;
所述供体DNA的序列如SEQ ID NO.2所示:
Oligo:
CCGTCTTCATGGCCTTGCCGTACACCGAGGTCTCAGGGACAC TGTGGCAGATCCAGATGCTCTTCAACTCCTTCCAGGTGTGCGGCC TGCCTGGGGTTCTGGGGAGTGCACGGGGTGGGGC。
3.根据权利要求1所述的一种原发性牙齿萌出障碍的PTH1R基因点突变小鼠模型的构建方法,其特征在于:所述gRNA、供体DNA和Cas9的混合物中,gRNA的浓度为2pmol/μl,供体DNA的浓度为100ng/μl,Cas9的浓度为30ng/μl。
4.根据权利要求1所述的一种原发性牙齿萌出障碍的PTH1R基因点突变小鼠模型的构建方法,其特征在于:所述基因型鉴定或筛选步骤中,繁育的小鼠出生后1-2周剪尾并提取基因组DNA,通过PCR反应,将得到的PCR产物进行测序、基因型鉴定或筛选;将PCR扩增产物进行测序,得到小鼠的基因型。
5.根据权利要求4所述的一种原发性牙齿萌出障碍的PTH1R基因点突变小鼠模型的构建方法,其特征在于:所述提取基因组DNA过程中使用TaKaRaMiniBEST通用基因组DNA提取试剂盒(Ver.5.0_CodeNo.9765)获得高纯度的基因组DNA。
6.根据权利要求4所述的一种原发性牙齿萌出障碍的PTH1R基因点突变小鼠模型的构建方法,其特征在于:所述PCR反应引物为:
正向引物(F1)的核苷酸序列如SEQ ID NO.3所示:5'-AGGAGAAGCGACTGTTGTGAGG-3';
反向引物(R1)的核苷酸序列如SEQ ID NO.4所示:5'-CCATTGCAGAAAGTATATGATG-3'。
7.根据权利要求4所述的一种原发性牙齿萌出障碍的PTH1R基因点突变小鼠模型的构建方法,其特征在于:所述PCR反应所使用的Taq DNA聚合酶是Green Taq Mix(VazymeP131)。
8.权利要求1-7任意一项所述的PTH1R基因点突变小鼠模型的构建方法构建得到的动物模型在制备研究原发性牙齿萌出障碍疾病的产品中的用途。
9.权利要求1-7任意一项所述的PTH1R基因点突变小鼠模型的构建方法构建得到的动物模型在验证治疗原发性牙齿萌出障碍药物的药效中的应用。
10.权利要求1-7任意一项所述的PTH1R基因点突变小鼠模型的构建方法构建得到的动物模型在评估牙、骨发育及其他基因富集组织器官表型分析中的应用,或者在评估颌骨骨髓间充质干细胞增殖、凋亡及成骨的影响研究中的应用。
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