CN108893495B - 一种Pdzd7基因突变动物模型的构建方法 - Google Patents
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Abstract
本发明公开了一种基于CRISPR/Cas9技术的Pdzd7基因突变动物模型的构建方法,步骤是(1)设计并选择合适的靶向小鼠Pdzd7基因的Guide RNA;(2)体外合成Cas9 mRNA和Guide RNA;(3)将Guide RNA和Cas9 mRNA等摩尔比共同配制成注射样品对受精卵注射;(4)对获得的F0代小鼠进行鉴定;(5)阳性F0代小鼠传代及F1代小鼠的获得与鉴定。本方法可以成功获得与人类中p.C732LfsX18致病突变相近的Pdzd7基因突变小鼠模型,为研究人类中PDZD7致病突变的相关因素和诊断治疗提供了基础。
Description
技术领域
本发明属于利用基因修饰技术制作基因突变动物模型技术领域,具体涉及一种基于CRISPR/Cas9技术的Pdzd7基因突变动物模型的构建方法。
背景技术
CRISPR/Cas(Clustered Regularly Interspaced Shot Palindromic repeats/CRISPR-associated)***是原核生物中一种抵抗外源基因侵入的获得性免疫***,在细菌和古生菌中CRISPR***整合侵入宿主的噬菌体或者质粒DNA的片段到CRISPR位点,然后通过相应的CRISPR RNAs(cr RNAs)引导Cas核酸内切酶对外源DNA序列进行切割,从而抵抗病毒或者噬菌体的入侵。CRISPR/Cas***分为3种类型(TypeⅠ、TypeⅡ和TypeⅢ),其中TypeⅡ***因其简单、高效以及功能多样化等优点,自2013在哺乳动物细胞中成功实现基因编辑后,现已被广泛用于多种模式生物的基因组修饰技术应用中。Ⅱ型***可以通过cr RNA的引导利用单个Cas9核酸酶对DNA靶位点进行精确充分地切割。CRISPR/Cas9***相关载体构建简单快速,便于操作,实验周期较短,且广泛适用于多个物种。在基因编辑的过程中,CRISPR/Cas9***需要一段特殊的引导RNA,即sgRNA(single guide RNA),sgRNA的序列选择限制性较小,只需要基因组序列中出现PAM(NGG)区即可。在sgRNA的引导下,Cas9蛋白可以对基因组实现定点切割,引起DNA双链断裂,继而引发细胞的自主修复,修复的过程可以是随机的片段缺失或***,也可以利用特定的模板修复,从而实现对基因组的永久性修饰。
Usher综合征(Usher syndrome,USH)是一种能够引起视觉和听觉双重感官障碍的遗传性疾病,在目前已知的超过50种的视力下降合并听力受损的综合征中,以Usher综合征最为常见,该病的全球发病率为1/6000~1/10000。USH为常染色体隐性遗传病,其特征表现为视网膜色素变性(retinitis pigmentosa,RP)和感音神经性耳聋(sensorineuralhearing loss,SNHL)。USH在临床上分为3种类型,都存在因视网膜色素变性而引起的进行性视力下降,区别在于听力和前庭功能的变现各不相同。其中USH1型最为严重,临床变现有先天性耳聋、严重的听力损失,因听力障碍导致的语言发育迟缓;此类患者还经常伴有前庭功能障碍,患儿学会独立行走的时间较同龄儿童晚。Usher2型患者听力损失较轻,为中到重度耳聋,但前庭功能正常。USH3型以渐进性听力损失为特点,伴随不同程度的前庭功能失调。此外,部分Usher综合征患者还可能伴有智力衰退、嗅觉丧失、癫痫等神经***病变。
PDZD7作为一个重要的耳聋基因,其编码蛋白是听觉感受细胞顶端听纤毛踝连接的组成成分,突变能够引起综合征型或非综合征型听力丧失。PDZD7基因有多个转录剪切本,编码不同的PDZD7蛋白亚型,其中长型PDZD7蛋白包含有三个重要的、能够介导蛋白质之间相互作用的结构域,即PDZ结构域,短型PDZD7只含有前两个PDZ结构域。目前常见的PDZD7突变大多影响长型PDZD7。在小鼠中模拟相关耳聋突变,构建Pdzd7基因突变小鼠模型,对PDZD7突变致聋机制进行深入探讨,并利用这一模型进行药物筛选,具有重要的临床意义。但检索发现目前虽有Pdzd7基因敲除小鼠,但没有仿真模拟人相关耳聋突变的、只影响长型PDZD7的小鼠模型,因此建立一种构建只影响长型PDZD7的基因突变动物模型的方法意义重大。
发明内容
本发明的目的是提供一种基于CRISPR/Cas9技术的Pdzd7基因突变动物模型的构建方法。
本发明所述基于CRISPR/Cas9技术的Pdzd7基因突变动物模型的构建方法,步骤是:
(1)在小鼠Pdzd7基因11号外显子上p.C728LfsX18致病突变位点附近设计合适的Guide RNA;其中引物Guide RNAtarget site 1的核苷酸序列如SEQ ID NO.1所示,引物Guide RNAtarget site 2的核苷酸序列如SEQ ID NO.2所示;
(2)体外合成Guide RNA和Cas9 mRNA;
(3)将合成后的Guide RNA和Cas9 mRNA等摩尔比配制成100 ul注射样品,将样品注射到取自C57BL/6J母鼠受精卵的雄原核中,培养恢复后移植入假孕受体小鼠,等待获得F0代小鼠;
(4)利用PCR反应体系对获得的F0代小鼠进行鉴定;
(5)将阳性F0代小鼠与野生型C57BL/6J交配获得F1代小鼠,即为Pdzd7基因突变小鼠模型。
上述基于CRISPR/Cas9技术的Pdzd7基因突变动物模型的构建方法中:步骤(2)所述Guide RNA和Cas9 mRNA体外合成由生工生物工程(上海)股份有限公司完成。
上述基于CRISPR/Cas9技术的Pdzd7基因突变动物模型的构建方法中:步骤(4)所述PCR反应体系的引物为Pdzd7-test-F3和Pdzd7-test-R3;其中Pdzd7-test-F3的核苷酸序列如SEQ ID NO.3所示,Pdzd7-test-R3的核苷酸序列如SEQ ID NO.4所示。
本发明公开的基于CRISPR/Cas9技术的Pdzd7基因突变动物模型的构建方法能够成功获得与人类中p.C732LfsX18致病突变相近的Pdzd7基因突变小鼠模型。方法中涉及的设计并挑选合适的靶向小鼠Pdzd7基因的Guide RNA,难点在于寻找728位Cysteine附近合适的sgRNA以实现能够模拟人类p.C732LfsX18突变的效果。本发明的构建方法为获得与人类Pdzd7基因p.C732LfsX18致病突变相近的小鼠模型提供了便利,为研究人类中PDZD7耳聋突变的相关因素和诊断治疗提供了基础。
附图说明
图1、Pdzd7基因突变小鼠构建策略图及基因鉴定引物设计。
图2、F0代阳性小鼠PCR产物测序结果比对。
具体实施方式
如下实施例使用的Taq酶及PCR相关试剂购自TaKaRa公司。化学试剂主要购自Sigma和上海化学试剂公司。胶回收Kit,质粒抽提Kit购于Qiagen公司。RNA合成自生工生物工程(上海)股份有限公司。引物合成自英潍捷基(上海)贸易有限公司。
实施例1:基于CRISPR/Cas9技术构建Pdzd7基因突变动物模型的构建方法通过以下步骤实现:
(1)靶向小鼠Pdzd7基因的Guide RNA的选择和设计
设计Pdzd7基因突变小鼠构建策略,如图1所示。
根据构建策略,在Pdzd7基因相应的位置设计合适的Guide RNA,其中Guide RNAtarget site1的核苷酸序列如SEQ ID NO.1所示,Guide RNA target site2的核苷酸序列如SEQ ID NO.2所示,具体序列如下:
sgRNA名称 | 序列 | PAM |
Guide RNA target site1 | ACAGGAGGTGGCTGGGGAGG | AGG |
Guide RNA target site2 | GAGGAGGTGCGCATGCGCCT | GCC |
(2)体外合成Guide RNA和Cas9 mRNA
Guide RNA和Cas9 mRNA由生工生物工程(上海)股份有限公司合成。
(3)受精卵显微注射制备F0代小鼠
1)单细胞受精卵的获得
小鼠超排:第一天,为C57BL/6J小鼠腹腔注射马绒毛膜***5IU/只,46-48小时后注射人绒毛膜***,注射完人绒毛膜***后将2只雌鼠与单放雄鼠1只合笼。第四天上午检栓,见栓的记为0.5天。
获取受精卵:将见栓0.5天后的小鼠脱颈处死,取出输卵管,在解剖镜下小心取出成团的卵子并用透明质酸酶消化,挑取形态饱满且胞质均匀的胚胎放置于M16培养液中培养。
2)显微注射受精卵
将挑选后的受精卵转移入M2条带中依次排列(30-50枚左右)。提前将合成后的Guide RNA和Cas9 mRNA等摩尔比共同配制成100ul注射样品,待注射管刺入受精卵后匀速将样品注入,见到胞质松散后迅速退针。注射结束后,将受精卵移入M16培养液中,并于37℃、5%二氧化碳培养箱中恢复0.5-1小时。将注射后的受精卵移植到E0.5天假孕小鼠体内。
移植后大约19-21天生出F0代小鼠。
(4)利用PCR反应体系对获得的F0代小鼠进行鉴定
1)F0代小鼠基因组制备
F0代小鼠出生14天剪取鼠尾0.3cm,加400ul裂解液及40ul蛋白酶K(10mg/ml),56℃消化过夜。离心取上清,两倍体积无水乙醇沉淀,75%乙醇洗涤,稍晾干后溶解于100ul纯水中,50℃烘箱助溶1小时。
2)F0代小鼠基因型鉴定
以制取的F0代小鼠基因组为模板使用TaKaRa Lataq扩增鉴定片段,引物为Pdzd7-test-F3和Pdzd7-test-R3,如图1所示,可扩增出1.5kb条带。其中,Pdzd7-test-F3的核苷酸序列如SEQ ID NO.3所示,Pdzd7-test-R3的核苷酸序列如SEQ ID NO.4所示。
Pdzd7-test-F3:5’-GGGGCGGGCGGGGACTT-3’
Pdzd7-test-R3:5’-CATGGGCTGCACCTTGGACTCG-3’
反应条件为98℃2min变性后,98℃10s,68℃60s,进行34个循环,68℃延伸2min,PCR体系参见TaKaRa说明书。
1%琼脂糖凝胶电泳后割胶回收1.5kb条带,分别连接pGEM-T载体(Promega),转化,每个平板挑取6个单克隆测序鉴定,引物为Pdzd7-test-F3。测序结果显示有2bp碱基缺失,如图2所示。
(5)将阳性F0代小鼠与野生型C57BL/6J交配获得F1代小鼠,筛选出的阳性F1代小鼠即为Pdzd7基因突变动物模型小鼠。
阳性F0代小鼠在8周龄左右性成熟后和野生型C57BL/6J回交进行配繁,得到F1小鼠。同F0代相似,待小鼠出生14天后,剪尾进行PCR鉴定,得到F1代杂合子。
筛选出的阳性F1代小鼠即为Pdzd7基因突变动物模型小鼠。
序列表
<110> 山东大学
<120> 一种Pdzd7基因突变动物模型的构建方法
<141> 2018-6-9
<160> 4
<170> PatentIn Version 3.5
<210> 1
<211> 20
<212> DNA
<213> 人工序列
<220>
<221> 小鼠
<222>(1)…(20)
<223> Guide RNA target site1的核苷酸序列
<400> 1
acaggaggtg gctggggagg 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列
<220>
<221> 小鼠
<222>(1)…(20)
<223> Guide RNA target site2的核苷酸序列
<400> 2
gaggaggtgc gcatgcgcct 20
<210> 3
<211> 27
<212> DNA
<213> 人工序列
<220>
<221> 小鼠
<222>(1)…(17)
<223> 引物Pdzd7-test-F3的核苷酸序列
<400> 3
ggggcgggcg gggactt 17
<210> 4
<211> 28
<212> DNA
<213> 人工序列
<220>
<221> 小鼠
<222>(1)…(22)
<223> 引物Pdzd7-test-R3的核苷酸序列
<400> 4
catgggctgc accttggact cg 22
Claims (1)
1.一种基于CRISPR/Cas9技术的Pdzd7基因突变动物模型的构建方法,步骤是:
(1)设计合适的Guide RNA;
(2)体外合成Guide RNA和Cas9 mRNA;
(3)将合成后Guide RNA和Cas9 mRNA等摩尔比配制成100μl注射样品,将样品注射到取自C57BL/6J母鼠受精卵的雄原核中,培养恢复后移植入假孕受体小鼠,等待获得F0代小鼠;
(4)利用PCR反应体系对获得的F0代小鼠进行鉴定;
(5)将阳性F0代小鼠与野生型C57BL/6J交配获得F1代小鼠,即为Pdzd7基因突变小鼠模型;
其特征在于:
步骤(1)所述设计合适的Guide RNA的策略是:在小鼠Pdzd7基因11号外显子上p.C728LfsX18致病突变位点附近设计合适的Guide RNA;其中引物Guide RNA targetsite1的核苷酸序列如SEQ ID NO.1所示,引物Guide RNA target site 2的核苷酸序列如SEQ ID NO.2所示;
步骤(4)所述PCR反应体系的引物为Pdzd7-test-F3和Pdzd7-test-R3;其中Pdzd7-test-F3的核苷酸序列如SEQ ID NO.3所示,Pdzd7-test-R3的核苷酸序列如SEQ ID NO.4所示。
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