CN113999873A - 一种基因修饰的非人动物的构建方法及其应用 - Google Patents
一种基因修饰的非人动物的构建方法及其应用 Download PDFInfo
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- CN113999873A CN113999873A CN202111658990.4A CN202111658990A CN113999873A CN 113999873 A CN113999873 A CN 113999873A CN 202111658990 A CN202111658990 A CN 202111658990A CN 113999873 A CN113999873 A CN 113999873A
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Abstract
本发明公开了一种基因修饰的非人动物的构建方法及其在生物医药领域的应用，具体的，将包含编码iCre‑ERT2的核苷酸序列敲入Adgra3基因，该编码iCre‑ERT2的核苷酸序列由内源性调控元件调控，获得脉络丛组织中表达Cre重组酶的工具非人动物。采用本申请构建的非人动物可以用于研究不同发育阶段脑脉络丛中Adgra3基因的表达情况及其可能参与的生物学功能，以及作为工具非人动物敲除特定基因，并进一步对该基因在脉络丛组织中的生物学功能、毒理学与药理学中可能的分子机制等进行精准研究。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种基因修饰的非人动物的构建方法及其在生物医药领域的应用。
背景技术
脉络丛（choroid plexus，CP）是由室管膜向脑室内陷后分化而形成的，在脑室内呈网状排列，根据其解剖定位不同，可以分为侧脑室脉络丛，第三脑室脉络丛及第四脑室脉络丛三种。脉络丛不仅是脑脊液(cerebrospinal fluid，CSF)的重要来源，而且可以通过其活性上皮所构成的血-脑脊液屏障(blood-cerebrospinal fluid barrier，BCB，见图1)将一些血源性物质有选择地向CSF内转运，以维持CSF环境的稳态，同时也阻止了一些存在于外周循环中的有害因子对脑组织可能造成的损伤。
在外源物质的毒性作用和药理作用中，脉络丛具有双重特性，既有神经保护作用（阻隔毒物进入脑组织和脑脊液）；又是一些神经毒物的靶组织（作为毒物蓄积的部位；或外源物质毒性作用可直接导致BCB结构和功能损伤）。从神经保护的角度来看，脉络丛具有大量的微绒毛和特定功能的蛋白受体/转运体，能够有选择地主动吸收或排出一些特定的物质，可作为一些有毒重金属（如铅、汞、镉等化合物）和其它外源化合物（如药物等）的贮存场所，在脉络丛组织内屏护并浓缩这些有毒重金属和有毒物质，阻止它们进入脑脊液，保持脑脊液成分的稳定，进而对中枢神经系统起到保护作用。
脉络丛在脑的发育、成熟、老化过程、内分泌调控以及一些神经退行性疾病的发病机理中也发挥着很重要的作用。另外，对于脉络丛在脑组织炎症、老化以及继发性神经毒性机制方面的研究均有所报道。但是，国内在这些方面的研究却很有限，主要集中于脉络丛的囊肿与肿瘤等方面，对于与脉络丛相关的毒理药理研究方面未引起足够的重视，有必要进一步加强。通过系统研究BCB在内、外源性化合物作用下的反应机制、在神经性损伤及损伤后修复过程中所发挥的作用及其调控机理等，有可能发现一些神经性毒物\神经损伤的早期诊断标志物与CNS特异性药物的转运蛋白等，推动CNS疾病诊治水平的发展。
粘附G蛋白偶联受体A3（Adhesion G Protein-coupled receptor A3，Adgra3），别名粘附G蛋白偶联受体125（Adhesion G protein-coupled receptors 125，GPR125），属于粘附G蛋白偶联受体（G protein-coupled receptors，GPCR）Ⅲ型，为一种跨膜蛋白。GPR125的胞外N端区域中所存在的免疫球蛋白（immuno-globulin，IG）样结构域具有调节细胞-细胞间粘附的作用；胞内C端的PDZ结合结构域（PDZ-binding domain）在有跨膜蛋白锚定时可以通过吸收hDlg而促进细胞的增殖。所以说GPR125在某些组织中（如脂肪组织）可以通过调节细胞-细胞以及细胞-基质间的粘附来调控细胞的存活与增殖等。另外，敲低GPR125后，体外研究发现细胞的聚集性减弱，体内研究发现骨髓肉瘤的发生率减少，说明grp125很可能是一个抑癌基因。GPR125还具有发育调控作用，如只存在于高度增生的成年精子发生祖细胞以及多能精原干细胞，而在细胞分化后却逐渐灭活消失。
Pickering等通过原位杂交、原位免疫组化共定位技术等发现GPR125在脑脉络丛组织中特异性表达，并且LPS诱导脑炎症后脉络丛与海马中的GPR125表达未改变，而在脑损伤后蛋白表达却增加显著（海马中GPR125含量增加延迟发生在脑损伤后1天），说明GPR125在机体应对脑损伤过程中发挥着重要的功能调控作用（Pickering C，Hagglund M，Szmydynger-Chodobska J，et al. The Adhesion GPCR GPR125 is specificallyexpressed in the choroid plexus and is upregulated following brain injury[J].BMC Neurosci.2008，9:97.）。
成簇规律间隔短回文重复序列系统(clusteredregularly interspaced shortpalindromic repeats，associated RNA guided endonuclease Cas，CRISPR/Cas)是细菌或古细菌在长期演化过程中形成的抵御外来遗传物质的一种获得性免疫防御机制。其中，II型CRISPR/Cas系统结构最为简单，可由向导RNA引导单一的Cas9蛋白特异性地切割目的DNA。利用CRISPR/Cas9基因编辑系统的优势，结合现有转基因动物技术，可以大幅度提高各种基因修饰动物的获得效率，缩短获得的时间，从而快捷有效地研究基因功能；同时，可建立包括灵长类疾病模型在内的多种动物疾病模型，促进生物和医学的发展。
然而，目前尚无在脑脉络丛组织实现组织特异性基因敲除的工具非人动物。
发明内容
本发明的目的是为了构建能够在脉络丛组织中表达Cre重组酶的非人动物，从而建成在脑脉络丛上皮组织进行特定基因敲除的工具，以此实现整体动物水平上对目的基因功能和作用机制的研究。具体的，利用CRISPR/Cas9的基因编辑技术将编码iCre-ERT2的核苷酸序列插入Adgra3基因启动子之后，获得基因修饰的非人动物。
本发明所采用的技术方案如下：
本发明的第一方面，提供了一种基因修饰的非人动物的构建方法，包括将外源核苷酸序列敲入Adgra3基因，所述的外源核苷酸序列包含编码Cre-ER的核苷酸序列，所述的外源核苷酸序列由非人动物Adgra3基因内源性调控元件调控，所述非人动物的脉络丛组织中表达Cre重组酶。
优选的，所述的外源核苷酸序列包括编码Cre-ERT的核苷酸序列。进一步优选的，包括编码Cre-ERT2的核苷酸序列。更进一步优选的，包括编码iCre-ERT2的核苷酸序列，其中，iCre-ERT2的氨基酸序列包含SEQ ID NO：17所示或者包含与SEQ ID NO：17具有80%同源性的氨基酸序列。编码iCre-ERT2的核苷酸序列包括SEQ ID NO：18。
优选的，所述编码iCre-ERT2的核苷酸序列与内源Adgra3基因直接连接或通过接头连接。所述的接头为现有技术常规使用的例如刚性接头、柔性接头、可裂解接头或无意义氨基酸等等。
在本发明的一个具体实施方式中，所述的接头为GSG-P2A序列。其氨基酸序列如SEQ ID NO：21所示；其核苷酸序列为如SEQ ID NO：22所示。
优选的，所述的外源核苷酸序列受他莫昔芬调控。
优选的，所述的外源核苷酸序列还包含编码接头的核苷酸序列，例如GSG-P2A。
优选的，所述的内源性调控元件包括但不限于启动子。
优选的，所述外源核苷酸序列的敲入不影响内源Adgra3的表达。在本申请的方案中外源插入的序列与内源Adgra3共表达。
优选的，所述的外源核苷酸序列敲入位置位于Adgra3基因的启动子下游，且终止密码子之前。
在本发明的一个具体实施方式中，所述的敲入位置位于Adgra3基因编码区（最后一个外显子编码区）之后，且终止密码子之前。
在本发明的一个具体实施方式中，获得的Adgra3基因座从5’至3’依次为Adgra3基因内源调控元件、Adgra3基因内源编码区、外源核苷酸序列、Adgra3基因内源终止密码子。
在本发明的一个具体实施方式中，获得的Adgra3基因座从5’至3’依次为Adgra3基因内源调控元件、Adgra3基因内源编码区、编码iCre-ERT2的核苷酸序列、Adgra3基因内源终止密码子。
在本发明的一个具体实施方式中，获得的Adgra3基因座从5’至3’依次为Adgra3基因内源调控元件、Adgra3基因内源编码区、编码GSG-P2A的核苷酸序列、编码iCre-ERT2的核苷酸序列、Adgra3基因内源终止密码子。
优选的，所述的脉络丛组织包括但不限于上皮组织和结缔组织，包括但不限于上皮细胞、室管膜细胞和巨噬细胞等等。
优选的，所述的构建方法包括但不限于利用胚胎干细胞的基因打靶技术、CRISPR/Cas9技术、锌指核酸酶技术、转录激活子样效应因子核酸酶技术、归巢核酸内切酶、RNAi技术、Cre/loxP技术、FLP/PLR技术或其他分子生物学技术。
优选的，所述的构建方法包括用sgRNA和/或打靶载体进行基因修饰的非人动物的构建。
进一步优选的，所述的sgRNA选自SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7或SEQ ID NO：8中的一种或两种以上。
进一步优选的，所述的打靶载体包含供体DNA序列、5’臂和3’臂；所述的供体DNA序列包含编码Cre-ER的核苷酸序列，优选的，包含编码Cre-ERT的核苷酸序列，进一步优选的，包括编码Cre-ERT2的核苷酸序列，更进一步优选的，包括编码iCre-ERT2的核苷酸序列，最为优选的，所述的供体DNA序列包含SEQ ID NO：18或者包含与SEQ ID NO：18具有80%同源性的核苷酸序列，所述的5’臂如SEQ ID NO：19所示，所述的3’臂如SEQ ID NO：20所示。
在本发明的一个具体实施方式中，所述的构建方法包括以下步骤：构建产生sgRNA的载体并获得sgRNA体外转录产物、构建包含外源核苷酸序列的打靶载体、将sgRNA体外转录产物和Cas9 mRNA以及打靶载体导入非人动物细胞，并进一步植入假孕母鼠的子宫，获得非人动物或其子代。其中，所述的非人动物细胞可以为非人动物的受精卵细胞、ES细胞、体细胞。
优选的，所述的非人动物为非人哺乳动物。进一步优选的，所述的非人动物可以为啮齿类动物，可以来自啮齿目、犬科、灵长目、偶蹄目。
优选的，所述的非人动物可以是免疫缺陷的。
优选的，所述的非人动物可以为大鼠、小鼠、狗、猴或猪。
在本发明的一个具体实施方式中，所述的非人动物为大鼠或小鼠。
本发明的第二方面，提供了一种上述构建方法获得的非人动物或其子代。
本发明的第三方面，提供了一种细胞、组织或器官，所述的细胞、组织或器官来源于上述非人动物或其子代。
本发明的第四方面，提供了一种细胞，所述细胞的Adgra3基因座敲入外源核苷酸序列，所述的外源核苷酸序列包含编码Cre-ER的核苷酸序列，所述的外源核苷酸序列由细胞内源Adgra3基因内源性调控元件调控，所述细胞中表达Cre重组酶。
优选的，所述的细胞来源于脉络从组织。
优选的，所述的细胞采用上述sgRNA和/或打靶载体构建获得。
本发明的第五方面，提供了一种靶向Adgra3基因的sgRNA，所述的sgRNA选自SEQID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ IDNO：7或SEQ ID NO：8中的一种或两种以上。
本发明的第六方面，提供了一种包含上述sgRNA的sgRNA寡核酸，所述的sgRNA寡核酸的底链序列如SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15或SEQ ID NO：16。
本发明的第七方面，提供了一种sgRNA载体，所述的载体产生上述的sgRNA。
优选的，所述的sgRNA载体还包括T7启动子及sgRNAscaffold的片段DNA。
本发明的第八方面，提供了一种打靶载体，所述的打靶载体包含供体DNA序列、5’臂和3’臂。
所述的供体DNA序列包含编码Cre-ER的核苷酸序列，优选的，包含编码Cre-ERT的核苷酸序列，进一步优选的，包括编码Cre-ERT2的核苷酸序列，更进一步优选的，包括编码iCre-ERT2的核苷酸序列，最为优选的，所述的供体DNA序列包含SEQ ID NO：18或者包含与SEQ ID NO：18具有80%同源性的核苷酸序列，所述的5’臂如SEQ ID NO：19所示，所述的3’臂如SEQ ID NO：20所示。
本发明的第九方面，提供了一种包含上述sgRNA、sgRNA载体或者打靶载体的细胞。
本发明的第十方面，提供了一种上述sgRNA、sgRNA载体、打靶载体或包含上述sgRNA、sgRNA载体或者打靶载体的细胞在Adgra3基因编辑中的应用。优选包括构建Adgra3基因编辑非人动物或者Adgra3基因编辑细胞中的应用。更进一步优选包括敲除、敲入、人源化等等非人动物或细胞的构建。
本发明的第十一方面，提供了上述构建方法获得的非人动物或其子代、上述细胞或者上述非人动物的应用，所述的应用包括但不限于在构建基因编辑动物模型中的应用，或者在研究Adgra3基因在脉络丛中的表达情况的应用。
进一步的，用于研究脉络丛组织在内、外源性化合物所致的脑损伤过程中的所发挥的作用及其分子机制。
进一步的，用于精准研究兴趣基因在脉络丛组织中的生物学功能、毒理学与药理学中可能的分子机制中的应用。
进一步的，用于研究脉络丛组织在内、外源性化合物所致的脑损伤过程中的所发挥的作用及其分子机制、精准研究兴趣基因在脉络丛组织中的生物学功能、毒理学与药理学中可能的分子机制中的应用。
优选的，用于在不同发育阶段脑脉络丛中Adgra3基因的表达情况及其可能参与的生物学功能的研究。
优选的，用于研究脉络丛组织参与的疾病。所述的脉络丛组织参与的疾病包括但不限于脑损伤疾病、肿瘤、囊肿、内分泌调控、神经退行性疾病、脑组织炎症或老化等等。
优选的，所述应用不是疾病的诊断和治疗方法。所述细胞、组织或器官不能发育为个体。
采用本发明所述的构建方法成功获得了脉络丛组织中特异性表达Cre重组酶的基因修饰的非人动物，而且应用该非人动物与Flox非人动物杂交成功获得的相应基因敲除的非人动物，可以进一步用于研究脉络丛组织在内、外源性化合物所致的脑损伤过程中的所发挥的作用及其分子机制，用于对敲除的相应基因在脉络丛组织中的生物学功能、毒理学与药理学中可能的分子机制等进行精准研究，用于研究不同发育阶段脉络丛中Adgra3基因的表达情况及其可能参与的生物学功能等等。本发明所述的Cre是指Cre(CyclizationRecombination Enzyme）重组酶，来源于噬菌体P1的一种酶蛋白，分子量约38kDa。它可以识别催化两个LoxP位点之间发生同源重组，从而造成DNA的缺失、易位等现象。
本发明所述的Cre-ER是指将人雌激素受体（estrogen receptor，简称ER）的配体结合区（LBD）与Cre重组酶相融合后，形成的定位于胞浆中的融合蛋白（Cre-ER），只有在激素诱导后，融合的Cre蛋白才会发生构象变化从锚定蛋白HSP90上解离下来，进入细胞核，识别loxP位点并发生切割。
本发明所述的Cre-ERT，是指在ER的配体结合区做一个点突变（G521R），获得的只响应外源的人工合成雌激素，例如他莫昔芬（Tamoxifen）的诱导Cre-ERT。
本发明所述的Cre-ERT2，为在Cre-ERT的基础上，在ER的LBD中增加3个点突变：C400V/M453A/L544A，得到的突变体就是Cre-ERT2。Cre-ERT2对他莫昔芬代谢产物4-OHT的敏感性更强。
本发明所述的iCre-ERT2，是在Cre-ERT2的基础上，进一步对Cre重组酶进行改进，旨在改善在哺乳动物体内的表达并减少表观遗传沉默的机会，具体参考文献Shimshek DR,et al. Codon-improved Cre recombinase (iCre) expression in the mouse.Genesis. 2002。
本发明所述的“基因座”广义上讲代表基因在染色体上所占的位置，狭义上讲代表某一基因上的一段DNA片段，即可以是一个基因也可以是一个基因的一部分。
本发明所述的“外源核苷酸序列”包含天然的或经过修饰的核糖核苷酸序列、脱氧核糖核苷酸序列。优选为DNA、cDNA、pre-mRNA、mRNA、rRNA、hnRNA、miRNAs、scRNA、snRNA、siRNA、sgRNA、tRNA。
本发明所述“同源性”，是指在使用蛋白序列或核苷酸序列的方面，本领域技术人员可以根据实际工作需要对序列进行调整，使使用序列与现有技术获得的序列相比，具有（包括但不限于）1%，2%，3%，4%，5%，6%，7%，8%，9%，10%，11%，12%，13%，14%，15%，16%，17%，18%，19%，20%，21%，22%，23%，24%，25%，26%，27%，28%，29%，30%，31%，32%，33%，34%，35%，36%，37%，38%，39%，40%，41%，42%，43%，44%，45%，46%，47%，48%，49%，50%，51%，52%，53%，54%，55%，56%，57%，58%，59%，60%，70%，80%，81%，82%，83%，84%，85%，86%，87%，88%，89%，90%，91%，92%，93%，94%，95%，96%，97%，98%，99%，99.1%，99.2%，99.3%，99.4%，99.5%，99.6%，99.7%，99.8%，99.9%的同一性。
除非特别说明，本发明的实践将采取细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术。这些技术在以下文献中进行了详细的解释。例如：Molecular Cloning A Laboratory Manual，2ndEd.，ed. By Sambrook，FritschandManiatis (Cold Spring Harbor Laboratory Press：1989)；DNA Cloning，Volumes I and II (D.N.Glovered.，1985)；Oligonucleotide Synthesis (M.J.Gaited.，1984)；Mullisetal. U.S. Pat.No.4，683，195；Nucleic Acid Hybridization(B.D.Hames& S.J.Higginseds.1984)；Transcription And Translation (B.D.Hames&S.J.Higginseds.1984)；Culture Of Animal Cells (R.I.Freshney，AlanR.Liss，Inc.，1987)；Immobilized Cells And Enzymes (IRL Press，1986)；B.Perbal，A PracticalGuide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY (J.Abelsonand M.Simon，eds.inchief，Academic Press，Inc.，New York)，specifically，Vols. 154and 155 (Wuetal.eds.) and Vol.185，″Gene Expression Technology″ (D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells (J.H.Miller and M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker，eds.，Academic Press，London，1987)；HandbookOf Experimental Immunology，Volumes V (D.M.Weir and C.C.Blackwell，eds.，1986)；and Manipulating the Mouse Embryo，(Cold Spring Harbor Laboratory Press，ColdSpring Harbor，N.Y.，1986)。
以上只是概括了本发明的一些方面，不是也不应该认为是在任何方面限制本发明。
本说明书提到的所有专利和出版物都是通过参考文献作为整体而引入本发明的。本领域的技术人员应认识到，对本发明可作某些改变并不偏离本发明的构思或范围。
下面的实施例进一步详细说明本发明，不能认为是限制本发明或本发明所说明的具体方法的范围。
附图说明
图1：血-脑屏障与血-脑脊液屏障的结构示意图（摘自Ghersi-Egea J，2002）；
图2：编码iCre-ERT2的核苷酸序列敲入大鼠Adgra3基因的技术路线图；
图3：F1代鼠尾DNA的Southern Blot检测结果，其中，WT为野生型对照，1EN5-18、1EN5-23、1EN5-26、1EN5-33、1EN5-34、1EN5-39、1EN5-40、1EN5-41、1EN5-44、1EN5-53及1EN5-56为鼠尾编号；
图4：PHB1条件性敲除大鼠鼠尾DNA的Southern blot检测结果，其中，WT为野生型对照；
图5：sgRNA相对活性检测结果，其中，Con.为空白对照，PC为阳性对照；
图6：打靶策略、打靶载体及探针、酶切位点设计；
图7A：F0代大鼠鼠尾鉴定结果，具体为采用引物EGE-WNN-005-L-GT-F/EGE-WNN-005-L-GT-R扩增的结果；
图7B：F0代大鼠鼠尾鉴定结果，具体为采用引物EGE-WNN-005-R-GT-F/EGE-WNN-005-R-GT-R扩增的结果；
图8A：F1代大鼠鼠尾鉴定结果，具体为采用引物EGE-WNN-005-L-GT-F/EGE-WNN-005-L-GT-R扩增的结果；
图8B：F1代大鼠鼠尾鉴定结果，具体为引物EGE-WNN-005-R-GT-F/EGE-WNN-005-R-GT-R扩增的结果。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或者制造商建议的条件进行。所用试剂或仪器未注明厂商者，均为可以通过市售购买获得的常规产品。
实施例1 Adgra3-iCreERT2模式大鼠的构建
本实施例以大鼠ENSRNOT00000036646.6转录本为例，将iCre-ERT2插入Adgra3基因中，使得Adgra3与iCre-ERT2共表达，构建Adgra3-iCreERT2模式大鼠。具体包括将P2A-iCreERT2元件插入Adgra3基因24号外显子与3’UTR之间的区域，其中，P2A-iCreERT2元件包括GSG-P2A（其氨基酸序列如SEQ ID NO：21所示，核苷酸序列如SEQ ID NO：22所示）与iCre-ERT2（其氨基酸序列如SEQ ID NO：17所示，核苷酸序列如SEQ ID NO：18所示）。Adgra3-iCreERT2模式大鼠的构建路线图如图2所示。步骤如下：
1）大鼠基因组靶序列的扩增和测序，不同品系，靶基因序列可能有所差异。为了保证所设计Cas9/sgRNA的效率，首先需要对SD大鼠尾靶位点序列进行PCR 扩增并测序验证，以保证sgRNA识别序列与SD鼠尾DNA 序列完全一致。PCR引物如表1所示。
表1.sgRNA靶位点扩增和测序引物
2）sgRNA的设计及活性检测
首先根据靶位点序列设计sgRNA序列（参见表2）。
表2. sgRNA序列
接下来，合成sgRNA寡核苷酸链（参见表3）。
表3.sgRNA寡核苷酸底链
利用百奥赛图自主开发的检测试剂盒UCATM对sgRNA/Cas9进行活性检测，结果见图5，综合sgRNA特异性以及活性，选择sgRNA3（sgRNA3的上下游序列见表4）进行后续实验。
表4.T7-sgRNA oligo
3）打靶载体的构建
根据插入位点的核苷酸序列，设计构建打靶载体，其包括待插入的编码iCreERT2的序列（SEQ ID NO.18），及与内源Adgra3基因核苷酸序列同源的5’臂（SEQ ID NO.19）和3’臂（SEQ ID NO.20），打靶策略及打靶载体见图6所示。并通过酶切鉴定和测序，确认打靶载体构建完成。
4）将sgRNA3连入带T7启动子质粒载体上并进行体外转录，得到进行显微注射的RNA。
5）获取大鼠受精卵，并原核注射sgRNA体外转录产物、Cas9 mRNA、打靶载体，将注射后的受精卵植入假孕母鼠子宫，获得F0代大鼠。
F0代大鼠鼠尾基因型鉴定，鉴定引物设计见表6-7，结果如图7A和图7B所示。通过PCR和测序结果表明，EN5-3、EN5-20、EN5-31、EN5-75和EN5-114为阳性F0大鼠。
将鉴定为阳性的EN5-3、EN5-20、EN5-31、EN5-75和EN5-114的F0代大鼠与野生型交配获得F1代杂合子大鼠。鼠尾鉴定引物同F0引物，结果如图8A和图8B所示。通过PCR鉴定与测序结果，表明1EN5-18、1EN5-23、EN5-26、1EN5-33、1EN5-34、1EN5-39、1EN5-40、1EN5-41、1EN5-44和1EN5-53为F1阳性大鼠。
用Junction PCR以及Southern杂交方法对F1代杂合子大鼠进行基因型鉴定。Southern探针设计见图6，利用BglII和AseI作为Southern blot酶切位点。5’Probe用于检测是否发生正确重组，若发生正确重组，将会出现野生型和突变型两个条带；若未正确重组，将只会出现野生型条带。iCre Probe用于检测是否含有随机插入，若发生正确重组，将只会出现突变型一条带；若存在随机插入，将会出现多条带。Southern结果如图3所示，1EN5-18、1EN5-23、EN5-26、1EN5-33、1EN5-34、1EN5-39、1EN5-40、1EN5-41、1EN5-44和1EN5-53均为没有随机插入的F1代阳性大鼠。构建探针所用引物见表5。
表5. 探针扩增所用引物
表6.基因型鉴定引物
表7. 基因型鉴定引物
实施例2
用以实施例1的方法获得的Adgra3-iCreERT2模式大鼠构建PHB1基因条件性敲除大鼠。步骤如下：
在大鼠PHB1基因的3号内含子和5号内含子分别插入LoxP序列获得flox大鼠。然后将其与实施例1获得的Adgra3-iCreERT2模式大鼠进行杂交，获得PHB1被条件性敲除的大鼠。
对PHB1条件性敲除大鼠进行Junction PCR以及Southern基因型检测，构建探针所用引物如表8所示。
表8.构建探针所用引物
Southern结果显示1EK33-21、1EK33-28、1EK33-38和1EK33-41为F1代阳性大鼠，并且均没有随机插入（参见图4）。
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tccatggagc acccagtgaa gctactgttt gctcctaact tgctcttgga caggaaccag 1440
ggaaaatgtg tagagggcat ggtggagatc ttcgacatgc tgctggctac atcatctcgg 1500
ttccgcatga tgaatctgca gggagaggag tttgtgtgcc tcaaatctat tattttgctt 1560
aattctggag tgtacacatt tctgtccagc accctgaagt ctctggaaga gaaggaccat 1620
atccaccgag tcctggacaa gatcacagac actttgatcc acctgatggc caaggcaggc 1680
ctgaccctgc agcagcagca ccagcggctg gcccagctcc tcctcatcct ctcccacatc 1740
aggcacatga gtaacaaagg catggagcat ctgtacagca tgaagtgcaa gaacgtggtg 1800
cccctctatg acctgctgct ggaggcggcg gacgcccacc gcctacatgc gcccactagc 1860
cgtggagggg catccgtgga ggagacggac caaagccact tggccactgc gggctctact 1920
tcatcgcatt ccttgcaaaa gtattacatc acgggggagg cagagggttt ccctgccaca 1980
gcttga 1986
<210> 19
<211> 1408
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
acatcaagaa ctatggcagc cggcccagtg caccctagtg agtaccccgt gttctctgta 60
cacctgagcg cactacctgg gtggtttgtg ggcacagttg acggcagtct cctaagcgct 120
ttattgaaat ccagagtgtc taagctagtt ccccatgttc tctcctagtt gctggatggc 180
ctgggaaccg tccttgggag ccttctacgg acctgccagc ttcatcactt ttgtaaactg 240
tatgtatttt ctaagcatat ttattcagtt gaaaagacac cctgagcgca aatacgagct 300
caaggagccg gcagaagagc agcagagatt ggcagctaat gaaaatggtg aaatcaatcc 360
tcaggactcc atgtctttgt ctctcatctc tacatccaca ctggaaaatg agcacagttt 420
tcagtcccag cttttggggg ccagccttac tctgcttttg tatatcacct tgtggatgtt 480
cggggccatg gcggtctctc tggattaccc tttagacttg gttttcagct tcttctttgg 540
agcctcttgt ctgagcttca gtgctttcat catggtgcac cactgcatca acagggagga 600
cgtgaggctt tcgtggatca tgatgtgctg cccgggacgg ggctcctact cggtgcaagt 660
caatgtccag cctcccacct caagtgcggc taacggagag gctccaaagt gcacgaatag 720
cagcgcagag tcttcatgca caaacaaaag cgcatcgagc ttcaaaaact cttcccaggg 780
ctgcaagcta acgaacctgc aggctgctgc agcacagtac cacagcaatg ccttacctgt 840
gagtgccaca cctcagcttg ataacagcct gaccgaacac tcgatggata acgatattaa 900
aatgcatgtg gctcctttag acgtgcagtt tcgagcaaac atgcatccaa gccgccatca 960
taaaaaccga agtaaaggac accgggccag cagactcacg gtcctgcgag agtatgccta 1020
tgatgtcccc acgagtgtgg aaggcagcgg gcagagcggc tcacctaaga gccggccagg 1080
cagcagtgaa ggacattcga ggagtcggag agcttacctg gcctatagag agaggcagtg 1140
caacccgccc caacaagaca gcagtgatgc tggtagcaca cttcccaaaa gtggccgaaa 1200
tgttgaaaag cctctttcaa ctagtagtaa gaaagatgcg ccaaggaagc cagctgcagc 1260
tgaccttgaa agccagcaga agtcgtatgg cctgaactta gctgttcaga atggaccagt 1320
taaaagcaat gggcaggaag gacccctgct agctactgat attactggca atattaggac 1380
tggactctgg aaacatgaaa caactgtg 1408
<210> 20
<211> 1354
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
cactgcgagg gcttccaaga cagggtgaaa ctgtgacact cacattcctt taagctatga 60
actcttagaa acaaactgtt tacagccacc ccagggatac aaaacagttc tgaatattct 120
tgtgagtttt gggacttact tattttatat tcctaaattg cccttcccca gaggttaaaa 180
acatgtttta aacattgttt tacttatcaa agtaccaata agatattttg gaagttgaaa 240
atataatttc ttagaatctg ttatatctac ttaacatttg aggtttgtat ttaataaaat 300
aaatagaagt ttgtcattgt gctggtggtt tatgtttaca ctgtaaaaag catgtgagga 360
gctggtgaga tggctccatg ggttaaaggc cccgcctgcc aagcatcctg gtttgtcagt 420
tgtaaactga cctcatccct gggtggaagg agacacccct cataagtggt cctctggcct 480
gcatgtggct tccaccacac atacataaat acatacatac atacacacac acatatacat 540
acatatacat acatgcatgc atcatatgtg cacacataca tatacataca tcatacacat 600
acttgcatac atacatatac acaatataca tacttataca cacatgcata cattatgcat 660
gcatacacac atacatacaa atatacacat acatacacat acctcataca catatataca 720
tacatacaca tacatcatac atacatgcat gcattttgag agcatttcag ctgtatttta 780
ataaagtttg tgaaatggtg tttttcaagg tgaatcaagc ataaataaat catcagatgt 840
actcttggct taactctact gaatataaca tttgtgtcaa aggatttctg agttaatctc 900
acagagtcat ttttaaaaat ggaaatacca ttggaacctg ttgtgtaaat tgcatatttt 960
tgccatatcg ctttgatcct attgatgtta ttttccacag tttcatagct tttcagtagt 1020
ttatggttga gttaaagttg atcttcgctt cctacccctt ttctccttcc tttctcctac 1080
agggtctcat ataccttagg ctgaaccccc acttgctgta tacctgacta tgactcccat 1140
tctttctctt gctgaaatga cagatgtgta ccattaggtc cagtttacat agtgctggga 1200
cccaaactcg gggctttgtt catgctggtc aggtgtttta ctaaccgtgt cgcatccgta 1260
gccttgagtt taacttctta gaggttgttc ttgtgttctg cacaggtact gttctttgaa 1320
tacagagaag ctctcaatga agctaccctt cact 1354
<210> 21
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val
1 5 10 15
Glu Glu Asn Pro Gly Pro
20
<210> 22
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60
ggacct 66
<210> 23
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gcatcaagct tggtaccgat gccgaaatgt tgaaaagcct ctttc 45
<210> 24
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
acttaatcgt ggaggatgat cagtgtaaac ataaaccacc agcac 45
<210> 25
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
taggcaatat taggactggg ctg 23
<210> 26
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
aaaccagccc agtcctaata ttg 23
<210> 27
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
cgtgatgagc ctcaacttct ccctg 25
<210> 28
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
cctttcacct ctccaagtcc acacc 25
<210> 29
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
ccttctgact ccaatgctgt gtc 23
<210> 30
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
atctctgccc agagtcatcc ttg 23
<210> 31
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
taagaaagat gcgccaagga agcca 25
<210> 32
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
acctctgggg aagggcaatt tagga 25
<210> 33
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
taagaaagat gcgccaagga agcca 25
<210> 34
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
ggcttgcagg tacaggaggt ag 22
<210> 35
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
tctatgacct gctgctggag 20
<210> 36
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
acctctgggg aagggcaatt tagga 25
<210> 37
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
gccattgtcc ctcctaggtt ctacc 25
<210> 38
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ctgcatgatt tcagggatgg acac 24
<210> 39
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
caagaccctg gtgtccacag 20
<210> 40
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
acgactgaag tgtactttgg ctgag 25
<210> 41
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
taaacaacgg gaacatgtct gaggg 25
<210> 42
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
gcatgtgtgg acgaagggag ataat 25
<210> 43
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
gagcgtccaa ccagatggca 20
<210> 44
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
catacaccag ggagaacagg c 21
<210> 45
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
ctgaggaggc tgagagaaag g 21
<210> 46
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
cttgagaggc tggctcacac 20
Claims (10)
1.一种基因修饰的非人动物的构建方法，其特征在于，包括将外源核苷酸序列敲入Adgra3基因，所述的外源核苷酸序列包含编码iCre-ERT2的核苷酸序列，所述的外源核苷酸序列由非人动物Adgra3基因内源性调控元件调控，所述非人动物的脉络丛组织中表达Cre重组酶。
2.权利要求1所述的构建方法，其特征在于，所述的iCre-ERT2的氨基酸序列包含SEQID NO：17，编码iCre-ERT2的核苷酸序列包括SEQ ID NO：18。
3.权利要求1所述的构建方法，其特征在于，所述编码iCre-ERT2的核苷酸序列与内源Adgra3基因直接连接或通过接头连接。
4.权利要求1所述的构建方法，其特征在于，所述的外源核苷酸序列敲入位置位于Adgra3基因的启动子下游，且终止密码子之前。
5.权利要求4所述的构建方法，其特征在于，包括用sgRNA和/或打靶载体进行基因修饰的非人动物的构建；
所述的sgRNA选自SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7或SEQ ID NO：8中的一种或两种以上，
所述的打靶载体包含供体DNA序列、5’臂和3’臂；所述的供体DNA序列包含编码iCre-ERT2的核苷酸序列，所述的编码iCre-ERT2的核苷酸序列包含SEQ ID NO：18，所述的5’臂如SEQ ID NO：19所示，所述的3’臂如SEQ ID NO：20所示。
6.权利要求1-5任一所述的构建方法，其特征在于，包括以下步骤：构建产生sgRNA的载体并获得sgRNA体外转录产物、构建包含外源核苷酸序列的打靶载体、将sgRNA体外转录产物和Cas9 mRNA以及打靶载体导入非人动物细胞，并进一步植入假孕母鼠的子宫，获得非人动物或其子代。
7.权利要求1-5任一所述的构建方法，所述的非人动物为大鼠或小鼠。
8.一种靶向Adgra3基因的sgRNA，其特征在于，所述的sgRNA选自SEQ ID NO：1、SEQ IDNO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7或SEQ ID NO：8中的一种或两种以上。
9.一种打靶载体，其特征在于，所述的打靶载体包含供体DNA序列、5’臂和3’臂；
所述的供体DNA序列包含编码iCre-ERT2的核苷酸序列，所述的编码iCre-ERT2的核苷酸序列包含SEQ ID NO：18，所述的5’臂如SEQ ID NO：19所示，所述的3’臂如SEQ ID NO：20所示。
10.权利要求1-7任一所述的构建方法获得的非人动物或其子代在构建基因编辑动物模型中的应用，或者在研究Adgra3基因在脉络丛中的表达情况的应用。
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