CN113652448B - 构建Tp53基因敲除金黄叙利亚仓鼠模型的方法和应用 - Google Patents
构建Tp53基因敲除金黄叙利亚仓鼠模型的方法和应用 Download PDFInfo
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Abstract
本发明提供构建Tp53基因敲除金黄叙利亚仓鼠模型的方法和应用,涉及疾病动物模型及其制备技术领域,所述方法包括:设计仓鼠Tp53基因特异性打靶序列、制备Tp53的打靶质粒载体pX330‑sgRNA、将pX330‑sgRNA显微注射至仓鼠受精卵的细胞核中、然后移植入受体母仓鼠内,F0代仓鼠出生,经鉴定该方法成功构建了Tp53基因敲除金黄叙利亚仓鼠模型。本发明阐述了第一个敲除Tp53基因的金黄叙利亚仓鼠的癌症表型,与Tp53突变的人类癌症类似,在Tp53‑/‑仓鼠模型发现多种类型的肉瘤,包括血管肉瘤、骨肉瘤,及低频癌,组织包括肾、胰腺和肾上腺。采用本发明所述方法制备的仓鼠模型能够补充当前小鼠模型,有利于研究Tp53缺乏症导致的人类癌症是如何发生发展,并为新型疗法的研究提供有效动物模型。
Description
技术领域
本发明涉及疾病动物模型及其制备技术领域,具体地,涉及一种癌症动物模型的构建方法和应用,尤其是构建Tp53基因敲除金黄叙利亚仓鼠模型的方法和应用。
背景技术
编码p53蛋白的Tp53肿瘤抑制基因是人类癌症中最常见的突变基因,在人类癌症中发生中约占50%以上,其中包括所有主要组织的癌变,但在其他类型癌症中该基因活性被抑制,因此,p53被称为“基因组的守护者”。继承突变Tp53等位基因的人类会患利弗劳梅尼综合征(Li-Fraumeni Syndrome,LFS),其特征是一系列早发性癌症,包括绝经前乳腺癌、软组织和骨肉瘤、肺癌、胰腺癌、皮肤癌和肾上腺皮质癌,部分脑肿瘤和白血病等。大多数Tp53突变是功能丧失(LOF)或显性负功能增益(GOF)错义突变,主要集中在外显子4-9,包括p53蛋白的DNA结合域,这些错义突变与癌细胞运动、侵袭和转移有关,Tp53突变是癌细胞侵袭性的重要预后预测因子,并且其与较差的临床结果相关。另有研究报道,p53基因的低表达或缺失可能影响动脉粥样硬化的发生。
目前已经建立了多种p53(其基因在小鼠中称为Trp53)缺陷的小鼠模型,上世纪九十年代初,为了模拟最初LSF小鼠模型,Donehower等人和Jacks等人建立了两个纯合敲除(KO)模型(Trp53-/-模型),研究发现Trp53-/-LFS小鼠在大约4个月大时死于胸腺T细胞淋巴瘤,同时低频率发生B细胞淋巴瘤和肉瘤,尤其是血管肉瘤。值得注意的是,早期的LFS小鼠模型(包括Trp53-/-模型和Trp53+/-模型)并没有概况在人类LFS患者中观察到的肿瘤的谱图、频率或潜伏期。癌症在Trp53-/-LFS小鼠模型中非常罕见。Olive等人建立LFS杂合的LOF错义突变和GOF点突变小鼠模型,如结构突变体Trp53R172H和DNA接触突变体Trp53R270H。这些小鼠发生多种癌症,使其更接近于人类LFS患者中发现的癌症。然而,目前已经建立的Trp53突变小鼠模型并没有完全概括在人类LFS患者中观察到的所有癌症,这些表现差异可能是由于物种和小鼠品系差异造成的。金黄叙利亚仓鼠是常用的实验小动物,与小鼠相比体型更大,且生理学特征与人类更相似,是研究人类疾病相关基础和药物开发的常用动物模型。与大、小鼠相比,金黄叙利亚仓鼠更适合代谢性疾病、癌症、心血管疾病和感染性疾病等研究。尤其在肿瘤方面的研究,金黄叙利亚仓鼠解剖学、生理学和病理学更接近人类,其荷载肿瘤时间长且肿瘤生长体积更大。但是由于金黄叙利亚仓鼠基因组序列不完全、胚胎干细胞培养不成熟导致利用传统方法制备基因敲除金黄叙利亚仓鼠困难,极少有关于叙利亚仓鼠肿瘤模型开发的研究报道。
发明专利CN108690839A虽然公开了一种以SD大鼠为背景的Tp53基因敲除大鼠模型的构建方法,但是其解决的是现有技术中对p53基因敲除大鼠模型制备过程中存在的成本高、制备周期长、背景不纯的技术问题,针对该方法制备出的大鼠模型的癌症表型并未说明。因此,构建新的Tp53敲除动物模型对研究有关Tp53缺乏症导致人类癌症的发病机制及其成为测试新型疗法的新模型具有重要的临床意义。
发明内容
(一)解决的技术问题
针对现有技术的不足,本发明解决了现有Trp53缺陷小鼠模型不能够概括全部Tp53缺陷人类癌症中观察到的全部癌症表型的技术问题,本发明提供了构建Tp53基因敲除金黄叙利亚仓鼠模型的方法和应用,本发明所述方法制备的动物模型能够补充当前小鼠模型,有利于研究Tp53缺乏症导致的人类癌症是如何发生发展,并未新型疗法的研究提供有效动物模型。
(二)技术方案
为实现以上目的,本发明通过以下技术方案予以实现:
一方面,本发明提供了一种构建Tp53基因敲除仓鼠模型的方法,所述方法利用CRISPR/Cas9技术建立Tp53基因敲除的金黄叙利亚仓鼠,敲除序列如SEQ ID NO:1所示;所述方法包括以下步骤:
(1)设计仓鼠Tp53基因特异性打靶序列,选择第5外显子上序列作为CRISPR作用的靶点设计sgRNA;所述sgRNA序列如SEQ ID NO:2所示;
(2)利用pX330-U6-Chimeric_BB-CBh-hSpCas9质粒(以下简称pX330质粒)获得Tp53的打靶质粒载体pX330-sgRNA,所述pX330质粒购自Addgene公司,Addgene编号:42230;
(3)将所述步骤(2)制备得到的pX330-sgRNA注射至仓鼠受精卵细胞核中,然后移植入受体母仓鼠内,F0代仓鼠出生,鉴定。
优选地,所述pX330-sgRNA按照以下步骤制备得到:用Bbs I限制性内切酶,单酶切pX330质粒。将Bbs I酶和pX330质粒放在37℃水浴锅中酶切2小时,65℃酶失活15min,用1%琼脂糖凝胶电泳分离酶切产物,回收线性化pX330质粒;将退火后的Oligo sgRNA SEQ IDNO:2通过T4连接酶室温1.5h连接线性化pX330质粒;将2μL的连接溶液加入DH5α感受态细胞转化均匀涂LB培养板9,37℃孵育12-16h;挑取上述步骤单克隆放入相同抗性LB培养液中,37℃摇床孵育12-16h;利用GeneJET Plasmid Miniprep Kit进行质粒提取,提取质粒测序鉴定,所用测序引物序列为SEQ ID NO:3;将测序正确地质粒,进行过夜扩大培养。利用QIAGEN EndoFree Plasmid Kit进行质粒抽提,抽提DNA质粒加入无内毒素TE溶液。
优选地,步骤(3)中用于鉴定F0代基因型使用的引物为:上游引物如序列SEQ IDNO:4所示,下游引物如序列SEQ ID NO:5所示,PCR反应产物琼脂糖凝胶电泳检测特异的目的条带后送Sanger测序,对色谱峰图和序列分析确定突变的位置。
另一方面,本发明提供的构建Tp53基因敲除仓鼠模型的方法应用于制备癌症领域研究的动物模型。
(三)有益效果
本发明提供的构建Tp53基因敲除仓鼠模型的方法中采用的sgRNA序列具有高效、不易脱靶的特点,本发明阐述了第一个敲除TP53肿瘤抑制基因的仓鼠的癌症表型,该动物模型出现多种癌症类型,这些癌症类型类似于人类LFS患者中观察到的癌症,以及Tp53突变癌症患者中观察到的癌症。与Tp53突变的人类癌症类似,在Tp53-/-仓鼠模型发现了各种肉瘤,包括血管肉瘤、骨肉瘤,以及低频癌,组织包括肾、胰腺和肾上腺;另外,Tp53依赖性骨髓疾病的仓鼠模型可以提供有关这些疾病如何在人类中发展的重要见解。采用本发明所述方法制备的小鼠模型能够补充当前小鼠模型,有利于研究Tp53缺乏症导致的人类癌症是如何发生发展,并为新型疗法的研究提供有效动物模型。
附图说明
图1A为设计靶向仓鼠TP53外显子5的sgRNA/Cas9表达载体的结构示意图;图1B为F0仓鼠缺失21个碱基和插入1个碱基的2种突变类型DNA序列;图1C为野生型叙利亚仓鼠DNA峰图与TP53基因敲除叙利亚仓鼠DNA峰图,红框显示TP53基因组位点中1bp的插入位置;图1D为野生型(WT)的p53蛋白表达图。
图2为Tp53基因型的Kaplan-Meier生存图。
图3为所构建的仓鼠模型形成的癌症的H&E图,其中图3A,3B,3C,3F,3G分别为骨骼肌、心脏、脾和肾的侵入性淋巴瘤细胞,图3D&H为肝脏和真皮中的血管核瘤,图3E为真皮中的鳞状细胞癌。
图4为仓鼠肝组织切片髓过氧化物酶免疫组化照片。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
构建Tp53基因敲除仓鼠模型
1材料
动物:叙利亚仓鼠购买于Charles River(LVG叙利亚仓鼠,品系代码:049)为例。本实施例中动物模型的制备及数据分析等实验通过伦理委员会实验动物伦理审查。所有仓鼠在清洁级动物房标准饲养,温度22-23℃,湿度45%-65%,光照周期为14光照/10黑暗。标准啮齿动物饲料喂养,自由采食和饮水。
2通过CRISPR/Cas9技术建立TP53基因敲除的金黄叙利亚仓鼠
(1)设计仓鼠Tp53基因特异性打靶序列,利用体外转录技术获得相应sgRNA;所述sgRNA序列如SEQ ID NO:2所示(sgRNA旨在靶向仓鼠TP53基因第五个外显子内的位点特异性序列);
(2)利用pX330-U6-Chimeric_BB-CBh-hSpCas9质粒(以下简称pX330质粒)获得Tp53的打靶质粒载体pX330-sgRNA,所述pX330质粒购自Addgene公司,Addgene编号:42230;,具体制备步骤如下:用Bbs I限制性内切酶,单酶切pX330质粒。将Bbs I酶和pX330质粒放在37℃水浴锅中酶切2小时,65℃酶失活15min,用1%琼脂糖凝胶电泳分离酶切产物,回收线性化pX330质粒;将退火后的Oligo sgRNA SEQ ID NO:2通过T4连接酶室温1.5h连接线性化pX330质粒;将2μL的连接溶液加入DH5α感受态细胞转化均匀涂LB培养板9,37℃孵育12-16h;挑取上述步骤单克隆放入相同抗性LB培养液中,37℃摇床孵育12-16h;利用GeneJET Plasmid Miniprep Kit进行质粒提取,提取质粒测序鉴定,所用测序引物SEQ IDNO 3为5'-GACTATCATATGCTTACCGT-3';将测序正确地质粒,进行过夜扩大培养。利用QIAGENEndoFree Plasmid Kit进行质粒抽提,抽提DNA质粒加入无内毒素TE溶液。
(3)将步骤(2)制备的pX330-sgRNA通过显微注射至仓鼠受精卵细胞核,然后移植入受体母仓鼠内,F0代仓鼠出生,鉴定;具体步骤如下:以矿物油覆盖的M2培养基为注射介质,使用TE缓冲液将DNA注射液稀释至10ng/μl,进行受精卵原核注射。在37.5℃、10%CO2、5%O2和85%N2条件下,将注射的受精卵在HECM-9培养基中培养0.5小时。挑选15枚有活力的受精卵移植到假孕雌性仓鼠的输卵管。代孕仓鼠正常饲养,避免打扰。F0待仓鼠出生2周后,取幼崽仓鼠脚趾提取基因组DNA,PCR扩增目的片段,PCR引物序列分别为上游引物p53-F,序列为5'-CTA AAC CAA CTG GTG TGT AGA ACC CC-3'(即序列SEQ ID NO:4所示),下游引物P53-R,序列为5'-GCT CAT AGG GCA CCA CCA CA-3'(即序列SEQ ID NO:5所示),PCR反应产物琼脂糖凝胶电泳检测特异目的条带后送Sanger测序,对色谱峰图和序列分析确定突变的位置。对确定发生突变的仓鼠进行以进一步的序列验证,将上述确定突变的PCR产物链接T载体,感受态细胞转化,接种含氨苄的LB培养皿,37℃孵育14h,菌液送Sanger测序,对色谱峰图和序列分析碱基突变位置、数目、次数。分析将蛋白移码突变F0代仓鼠与野生型仓鼠杂交后获得F1代仓鼠,后代提取脚趾基因组DNA进行测序,选取插入1个碱基移码突变的仓鼠繁育。
3测试与方法
3.1组织病理学及免疫组织化学
仓鼠全身用福尔马林固定3天,然后储存在70%乙醇中。对肿瘤组织进行石蜡包埋处理,组织切片用苏木精和伊红(H&E)进行染色。免疫组织化学按标准步骤。抗体检测:柠檬酸低pH 6.0,30分钟;Abcam抗体:cat#ab9535,稀释1:50,30分钟;检测:Dako RabbitEnvision-30分钟;显色剂:DAB 5分钟。抗CD3抗体,DAKO/Agilent,Santa Clara,CA,美国;货号A0452。
3.2蛋白质印迹
将仓鼠耳组织在液氮中研磨,然后在裂解缓冲液(150mM NaCl、1.0%Triton X-100和50mM Tris,pH 8.0,以及蛋白酶抑制剂混合物(Sigma))中裂解。通过Pierce BCA蛋白质测定试剂盒(Thermo Scientific)对总蛋白质质量进行量化。组织裂解物在NuPAGE4–12%Bis-Tris凝胶(Life Technologies)上分离,然后电转移到PVDF膜(LifeTechnologies)。封闭和洗涤步骤使用WesternBreeze化学发光试剂盒(LifeTechnologies)进行,并严格遵循说明书步骤。将膜在室温下与一抗小鼠单克隆抗P53抗体(Abcam、PAb 240、ab26)一起在1%BSA/PBS中以1:500稀释液孵育1小时。洗涤后,将膜与碱性磷酸酶偶联的二抗(抗兔)孵育30分钟并发光。
3.3 Kaplan-Meier分析
TP53+/+动物在365天时被处死。TP53+/-和TP53-/-动物处于垂死或困境时立即处死。Kaplan-Meier分析用于可视化基因型之间的存活差异。使用对数秩检验和Bonferroni校正进行多重比较,确定基因型之间差异的显着性。P≤0.05表示具有显著性差异。
4结果与分析
4.1通过CRISPR/Cas9技术建立Tp53基因敲除的金黄叙利亚仓鼠
本发明设计了靶向仓鼠Tp53外显子5的sgRNA/Cas9表达载体(如图1A所示)。采用原核注射将10ng/μL的载体注射到金黄叙利亚仓鼠的受精卵的雄核中。
图1B所示亚克隆测序F0仓鼠,携带移码突变(1bp插入)、21bp缺失。为检测突变等位基因向F1代建立者的种系传递,选择该仓鼠建立繁殖集落,其携带1bp插入稳定传代。
图1C显示了野生型叙利亚仓鼠DNA峰图与Tp53基因敲除叙利亚仓鼠DNA峰图比较,红框显示Tp53基因组位点中1bp的插入位置。
采用WB法检测p53纯合(+1bp)成纤维细胞系和野生型成纤维细胞系的细胞裂解物,结果显示,p53纯合(+1bp)无p53抗体表达,野生型(WT)有p53蛋白表达(图1D),证明p53蛋白失活,Tp53基因敲除的金黄叙利亚仓鼠建模成功。
4.2 Tp53-/-和Tp53+/-金黄叙利亚仓鼠的存活率
图2显示了Tp53基因型的Kaplan-Meier生存图。Tp53-/-仓鼠平均存活至139天,而Tp53+/-仓鼠平均存活至286天。
4.3癌症类型和频率
表1为82只(52只Tp53-/-和30只Tp53+/-)突变仓鼠的病理分析,列出了观察到的各种类型的癌症及其频率。
表1癌症发病率和频率
P53-/-(N=52) | 发生次数 | 频率 | P53+/-(N=30) | 发生次数 | 频率 |
淋巴瘤 | 15 | 29% | 淋巴瘤 | 20 | 67% |
血管肉瘤 | 14 | 27% | 血管肉瘤 | 5 | 17% |
髓系白血病 | 11 | 21% | 髓系白血病 | 2 | 6% |
间变性肉瘤 | 9 | 17% | 间变性肉瘤 | 0 | 0 |
髓系增生 | 5 | 10% | 髓系增生 | 1 | 3% |
肾上腺皮质癌 | 4 | 8% | 肾上腺皮质癌 | 1 | 3% |
骨肉瘤 | 3 | 6% | 骨肉瘤 | 1 | 3% |
胰腺癌 | 2 | 4% | 胰腺癌 | 0 | 0 |
浆细胞瘤 | 1 | 2% | 浆细胞瘤 | 0 | 0 |
鳞状细胞癌 | 1 | 2% | 鳞状细胞癌 | 0 | 0 |
肾腺癌 | 0 | 0 | 肾腺癌 | 1 | 3% |
4.4癌症表型
组织病理学结果显示所构建的仓鼠模型形成癌症类型。图3显示了几种癌症的H&E,特别是骨骼肌、心脏、脾和肾的侵入性淋巴瘤细胞(图3A,B,C,F,G),肝脏和真皮中的血管核瘤(图3D&H)和真皮中的鳞状细胞癌(图3E)。总之,43%的突变仓鼠发生淋巴瘤,39%的仓鼠形成了一系列的肉瘤,血管肉瘤最常见的类型。
图4结果所示,突变仓鼠肝癌中观察到髓过氧化物酶(MPO)染黄色阳性(图4A&C)。提示淋巴瘤可能是T细胞淋巴瘤。
分别67%的Tp53+/-仓鼠和29%Tp53-/-仓鼠的发生淋巴瘤,TP53+/-仓鼠发生淋巴瘤频率较高;Tp53-/-仓鼠与Tp53+/-仓鼠的髓细胞白血病(和骨髓增殖)的频率显著增高。Tp53+/-仓鼠可以形成两种上皮癌,包括肾细胞癌和肾上腺皮质癌,而Tp53-/-仓鼠可产生6种上皮癌,包括肾上腺皮质癌、肾细胞癌、胰腺癌、口腔鳞状细胞癌等。
5讨论
随着CRISPR/CAS9技术在叙利亚仓鼠基因工程中的成功应用,叙利亚仓鼠成为研究人类疾病包括癌症的新型遗传模型。在这里,本发明阐述了第一个敲除Tp53肿瘤抑制基因的仓鼠的癌症表型。Tp53-/-仓鼠很早形成癌症,中位52天可以出现垂死。该动物模型出现多种癌症类型,这些癌症类型类似于人类LFS患者中观察到的癌症,以及Tp53突变癌症患者中观察到的癌症。与Tp53突变的人类癌症类似,在Tp53-/-仓鼠发现了各种肉瘤,尤其是血管肉瘤、骨肉瘤,以及低频癌肾、胰腺和肾上腺。
仓鼠和人类常见的另一种Tp53依赖性癌症是髓细胞白血病。Tp53-/-仓鼠展示了与髓细胞白血病一致的侵袭性肿瘤,发病率为16%。虽然Tp53突变的髓系白血病在LFS患者中不常见(白血病总体发生率仅为4%),但它们是LFS患者治疗相关的急性髓系白血病(AML)和骨髓增生异常综合征(MDS)中的一个非常严重的问题。在散发性人类癌症中,约10%的AMLS伴有Tp53突变,表现为异常急性AMLS,并影响化疗疗效。在非LFS患者中,Tp53突变也有约20%的MDS中观察到。此外,通过大规模测序、蛋白质组学和临床研究测量的p53功能障碍在人类AML中非常普遍,与Tp53突变状态无关,这是由p53抑制剂(如MDM2和MDM4)的失调所致。AML中Tp53的失调,无论是通过突变还是通过抑制野生型p53,都为AML新疗法的开发带来了治疗挑战和机遇。因此,Tp53依赖性骨髓疾病的仓鼠模型可以提供有关这些疾病如何在人类中发展的重要见解,并为测试新疗法提供模型。
将Tp53突变仓鼠的癌症表型与Trp53突变小鼠模型进行比较是很重要的。Tp53+/-仓鼠和Trp53+/-小鼠患癌症的潜伏期大约是纯合突变体年龄的两倍,癌症范围更有限。Trp53纯合突变小鼠死于T细胞淋巴瘤,偶尔出现肉瘤,没有癌。靶向Trp53+/-GOF点突变体小鼠,如Trp53R172H和Trp53R270H会发展出更广泛的癌症,这些癌症与TP53-/-仓鼠中发现的癌症相似。但是Trp53R270H/+小鼠也会发展成B细胞淋巴瘤。与仓鼠不同的是,Trp53缺陷的小鼠模型通常不会发展为AML、MDS或其他骨髓疾病。这种对骨髓疾病易感性的差异证明仓鼠可更好地模拟这些疾病。
以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
序列表
<110> 郑州大学
<120> 构建Tp53基因敲除金黄叙利亚仓鼠模型的方法和应用
<130> 2021.6.1
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1191
<212> DNA
<213> 人工合成()
<400> 1
atggaggagc cacagtcaga cctcagcatc gagctccctc tgagtcagga gacattttca 60
gacctgtgga aactacttcc tccaaacaat gttctgtcca ccttgccgtc ctctgattcc 120
attgaagaac tgttcctgtc cgagaatgtt gcaggctggc tagaagaccc aggtgaagct 180
ctccaagggt cggcggctgc ggcagcgccg gcggctcctg cagcagagga ccctgtagct 240
gagactcctg caccggtggc ctctgcgcca gccactccct ggcccctctc atcttctgtc 300
ccatcctata aaacctacca gggcgactat ggtttccgtc tgggcttcct gcactcgggg 360
acggccaaat ctgtcacatg cacgtactca ccttccctca ataagctgtt ctgccagctg 420
gcgaaaacat gccccgtgca gctgtgggtc agctccacac ctccacctgg cacccgtgtc 480
cgtgccatgg ccatctacaa gaagttacaa tacatgacgg aagttgtaag acgctgtccc 540
caccacgagc gctcctccga gagcgatggt ttggctcctc ctcagcatct tatccgagtg 600
gaaggaaata tgcatgccga atacctggat gacaagcaga cttttcggca cagtgtggtg 660
gtgccctatg agccacctga ggttggctct gactgtacca ccatccacta taactacatg 720
tgtaatagtt cctgcatggg gggcatgaac cggcggccta tcctcaccat catcacgctg 780
gaggacccca gtgggaacct gctgggacgg aacagctttg aggttcgtat ttgtgcctgc 840
cctgggagag accgtcgtac agaggaaaaa aatttccaaa agaagggaga accttgccca 900
gaactacccc caaagagtgc taaacgagca ttgcctacca acacaagctc ctctccccag 960
ccaaagagaa aaacacttga cggagaatat ttcaccctta agatccgtgg tcaagaacgc 1020
ttcaagatgt tccaagaatt gaatgaggcc ttggaactga aggatgcaca ggctttgaag 1080
gcgtcagagg acagtggtgc tcactccagc tacctgaagt ccaagaaggg ccagtctgcc 1140
tcccgtctta aaaaactaat gatcaagaga gaggggcctg actcggactg a 1191
<210> 2
<211> 20
<212> DNA
<213> 人工合成()
<400> 2
gtcagctcca cacctccacc 20
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<212> DNA
<213> 人工合成()
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gactatcata tgcttaccgt 20
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<212> DNA
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ctaaaccaac tggtgtgtag aacccc 26
<210> 5
<211> 20
<212> DNA
<213> 人工合成()
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gctcataggg caccaccaca 20
Claims (3)
1.构建Tp53基因敲除金黄叙利亚仓鼠模型的方法在制备癌症动物模型中的应用,其特征在于,所述癌症选自髓细胞白血病、浆细胞瘤、肾上腺皮质癌、肾细胞癌、胰腺癌、口腔鳞状细胞癌;所述方法利用CRISPR/Cas9技术建立Tp53基因敲除的金黄叙利亚仓鼠,所述Tp53基因序列如SEQ ID NO:1所示;所述方法包括以下步骤:
(1)设计仓鼠Tp53基因特异性打靶序列,选择第5外显子上序列作为CRISPR作用的靶点设计sgRNA,所述sgRNA序列如SEQ ID NO:2所示;
(2)利用pX330-U6-Chimeric_BB-CBh-hSpCas9质粒获得Tp53的打靶质粒载体pX330-sgRNA;
(3)将所述步骤(2)制备得到的pX330-sgRNA注射至仓鼠受精卵细胞核中,然后移植入受体母仓鼠内,F0代仓鼠出生,鉴定。
2.根据权利要求1所述的构建Tp53基因敲除金黄叙利亚仓鼠模型的方法在制备癌症动物模型中的应用,其特征在于,所述pX330-sgRNA按照以下步骤制备得到:
将Bbs I酶和pX330-U6-Chimeric_BB-CBh-hSpCas9质粒放在37℃水浴锅中酶切2小时,65℃酶失活15min,用1%琼脂糖凝胶电泳分离酶切产物,回收线性化质粒;将退火后的Oligo sgRNA通过T4连接酶室温1.5h连接所述线性化质粒;将2μL的连接溶液加入DH5α感受态细胞转化均匀涂LB培养板,37℃孵育12-16h;挑取单克隆接种至LB培养液,37℃摇床孵育12-16h;利用GeneJETPlasmid Miniprep Kit进行质粒提取,提取质粒测序鉴定,所用测序引物序列为SEQ IDNO:3;将测序正确的质粒,进行过夜扩大培养;利用QIAGEN EndoFreePlasmid Kit进行质粒抽提,抽提DNA质粒加入无内毒素TE溶液。
3.根据权利要求1所述的构建Tp53基因敲除金黄叙利亚仓鼠模型的方法在制备癌症动物模型中的应用,其特征在于,用于鉴定F0代基因型的引物为:上游引物如序列SEQ ID NO:4所示,下游引物如序列SEQ ID NO:5所示,PCR反应产物琼脂糖凝胶电泳检测特异的目的条带后送Sanger测序,对色谱峰图和序列分析确定突变的位置。
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