CN108642090A - 基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法及应用 - Google Patents
基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法及应用 Download PDFInfo
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Abstract
本发明涉及一种基于CRISPR/Cas9技术获得Nogo‑B敲除模式小鼠的方法及应用，获得Nogo‑B敲除模式小鼠的方法包括步骤：设计高效的识别特定基因EGE‑ZXH‑007的sgRNA；将sgRNA连入质粒载体上，然后进行体外转录，得到可进行显微注射的Cas9/sgRNA；将可进行显微注射的Cas9/sgRNA显微注射到小鼠受精卵中，然后对出生的小鼠进行基因型检测和鉴定，确认最终得到Nogo‑B敲除模式小鼠。本发明采用CRISPR/Case9技术制备Nogo‑B敲除模式小鼠，与传统基因敲除技术相比，更易于操作，效率更高，更容易得到纯合子突变，为进一步研究Nogo‑B的功能打下良好的基础。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法及应用。
背景技术
基因敲除是针对某个序列已知但功能未知的序列，改变生物的遗传基因，令特定的基因功能丧失作用，从而使部分功能被屏障，并可进一步对生物体造成影响，进而推测出该基因的生物学功能。
基因敲除技术是20世纪80年代发展起来的，是建立在基因同源重组技术基础以及胚胎干细胞技术基础上的一种新分子生物学技术。基因同源重组技术是指外源DNA与受体细胞基因组中序列相同或相近的基因发生同源重组，从而代替受体细胞基因组中的相同/相似的基因序列，整合入受体细胞的基因组中。胚胎干细胞是从着床前胚胎(孕3-5天)分离出的内细胞团细胞，它具有向各种组织细胞分化的多分化潜能，能在体外培养并保留发育的全能性，在体外进行遗传操作后，将它重新植回小鼠子宫，它能发育成胚胎的各种组织。
基因敲除动物模型是在活体动物上开展基因功能研究的重要工具。但是传统的基因敲除方法需要通过复杂的打靶载体构建、胚胎干细胞筛选、嵌合体小鼠选育等一系列步骤，不仅流程繁琐、对技术的要求很高，而且费用大，耗时较长，成功率受到多方面因素的限制。即使对于技术比较成熟的实验室，利用传统技术构建基因敲除小鼠一般也需要一年以上。
Nogo-B是网状蛋白家族4的重要成员，广泛表达于中枢神经系统及外周组织，其亚细胞定位于细胞内质网及细胞膜，Nogo-B主要参与血管损伤、组织修复、再生及炎症过程的调控，并可能在肿瘤细胞凋亡中也发挥重要作用。因此构建Nogo-B敲除模式小鼠，对于深入研究Nogo-B在多种疾病中的作用机制有重要的意义。
发明内容
针对现有技术中的缺陷，本发明目的在于提供一种基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法及应用，构建方法易于操作，效率高，更容易得到纯合子突变，为进一步研究Nogo-B的功能打下良好的基础。
为实现上述目的，本发明提供的技术方案为：
本发明提供了一种基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法，包括步骤：设计高效的识别特定基因EGE-ZXH-007的sgRNA；将sgRNA连入质粒载体上，然后进行体外转录，得到可进行显微注射的Cas9/sgRNA；将可进行显微注射的Cas9/sgRNA显微注射到小鼠受精卵中，然后对出生的小鼠进行基因型检测和鉴定，确认最终得到Nogo-B敲除模式小鼠。
优选地，基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法中，sgRNA的序列如SEQ ID NO.1和SEQ ID NO.2所示。
优选地，基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法中，质粒载体为带T7启动子的质粒载体。
优选地，基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法中，在将sgRNA连入质粒载体上之前，还包括Cas9/sgRNA构建和活性检测的步骤。
优选地，Cas9/sgRNA构建和活性检测具体包括：将sgRNA连入pCS载体，得到连接产物Cas9/sgRNA；将Cas9/sgRNA转化，然后测序验证正确；再对Cas9/sgRNA进行活性检测。
本发明还保护基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法获得的Nogo-B敲除模式小鼠在Nogo-B的功能研究中的应用。
本发明还保护一种获得Nogo-B敲除模式小鼠的试剂盒，试剂盒包括能识别特定基因EGE-ZXH-007的sgRNA。
优选地，获得Nogo-B敲除模式小鼠的试剂盒中，sgRNA的序列如SEQ ID NO.1和SEQID NO.2所示。
Nogo-B敲除模式小鼠的构建方法具体包括步骤：(1)分析EGE-ZXH-007基因的结构，该基因有3个主要转录本A、B、C，该设计要敲掉A、B两个转录本的第一个外显子，转录本C的第一个外显子位于下游，两条sgRNAs可以敲掉EGE-ZXH-007基因A、B两个转录本的第一个外显子约～2kb，基于CRISPR/Cas9技术制备模式小鼠；(2)Cas9/sgRNA设计及构建；(3)Cas9/sgRNA的活性检测；(4)sgRNA的RNA制备：将sgRNA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)Cas9/sgRNA的显微注射：将Cas9/sgRNA，显微注射到小鼠受精卵中，注射后F0小鼠出生；(6)F0代小鼠基因型检测；(7)F1代小鼠基因型鉴定。
CRISPR是细菌用来抵御病毒侵袭/躲避哺乳动物免疫反应的基因系统。利用RNA引导Cas9核酸酶可在多种细胞(包括iPS)的特定的基因组位点上进行切割，修饰。与传统基因敲除技术相比，CRISPR/Cas9更易于操作，效率更高，更容易得到纯合子突变。采用CRISPR/Case9技术制备Nogo-B敲除模式小鼠，为进一步研究Nogo-B的功能打下良好的基础。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
图1为本发明实施例中的EGE-ZXH-007基因的序列相关信息图；
图2为本发明实施例中的EGE-ZXH-007基因的转录本图；
图3为本发明实施例中的EGE-ZXH-007基因的结构域图；
图4为本发明实施例中的EGE-ZXH-007基因的调控区域图；
图5为本发明实施例中的打靶载体示意图；
图6为本发明实施例中的precut pCS质粒图谱图；
图7为本发明实施例中的Cas9/sgRNA的活性检测检查结果图；
图8为本发明实施例中的可进行显微注射的RNA的电泳图；
图9为本发明实施例中的F0代小鼠鼠尾基因型鉴定结果图；
图10为本发明实施例中的F1代小鼠基因型鉴定结果图；
图11为本发明实施例中的F1代小鼠基因型鉴定结果图；
图12为本发明实施例中的Nogo-B敲除小鼠肝脏Western Blot检测结果图。
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。以下实施例仅用于更加清楚地说明本发明的技术方案，因此只是作为示例，而不能以此来限制本发明的保护范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，数据为三次重复实验的平均值或平均值±标准差。本发明是在国家自然科学基金资助项目(项目批准号81570563)的支持下进行的。
实施例
EGE-ZXH-007基因在11号染色体正链上，全长约51.1kb。NCBI ID:68585(如图1所示)。EGE-ZXH-007基因有10个转录本，本设计参照转录本005(Rtn4-005)，Transcript ID：ENSMUST00000102843(如图2所示)。EGE-ZXH-007基因的结构域如图3所示。EGE-ZXH-007基因的调控区域如图4所示。打靶策略：分析EGE-ZXH-007基因的结构，该基因有3个主要转录本A、B、C，该设计要敲掉A、B两个转录本的第一个外显子，转录本C的第一个外显子位于下游，两条sgRNAs可以敲掉EGE-ZXH-007基因A、B两个转录本的第一个外显子约～2kb，基于CRISPR/Cas9技术制备模式小鼠。打靶载体示意图如图5所示。
下面具体阐述基于CRISPR/Cas9技术获得EGE-ZXH-007基因敲除模式小鼠的方法。
1.靶序列的测序确认
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C57BL/6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C57BL/6鼠尾DNA序列完全一致。PCR引物如下表1所示。
表1 PCR引物序列表
对C57BL/6鼠尾DNA进行PCR及测序，结果证明：C57BL/6鼠尾靶序列与Genebank和Ensembl所给序列完全一致。
2.Cas9/sgRNA设计及构建
2.1 Cas9/sgRNA的设计
基于sgRNA的设计原则，在靶位点区域共设计14条sgRNA，其对应的oligo序列如下表2所示。
表2 oligo序列表
2.2 Cas9/sgRNA质粒的构建
按照已设计sgRNA序列合成引物，通过退火聚合的方式连入pCS载体，连接产物转化后送样测序验证正确。
载体图谱(precut pCS质粒图谱)如图6所示。
3.Cas9/sgRNA的活性检测
对sgRNA的活性检测，根据检测结果综合选择EGE-ZXH-007-sgRNA2(对应SEQ IDNO.1)和EGE-ZXH-007-sgRNA10(对应SEQ ID NO.2)进行下一步实验。检测结果如图7所示。
4.sgRNA的RNA制备
将EGE-ZXH-007-sgRNA2和EGE-ZXH-007-sgRNA10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Cas9/sgRNA。
RNA电泳图如图8所示。
5.Cas9/sgRNA的显微注射
将Cas9/sgRNA，显微注射到小鼠受精卵中，注射后F0小鼠出生。
表3显微注射
6.F0代小鼠基因型检测
本发明使用Cas9/sgRNA注射受精卵的方法构建基因敲除小鼠。由于胚胎早期卵裂速度很快，因此得到的F0小鼠为嵌合体。故以F0小鼠鼠尾进行鉴定得到的F0基因型仅供参考，不能代表其一定为可遗传的基因突变型，可遗传的基因型需待F1小鼠鼠尾检测后确定。
6.1鉴定引物设计
引物设计原则如图5所示。
引物信息如下表4所示。
表4引物序列表
PCR的条件为：
6.2 F0代小鼠鼠尾基因型鉴定
部分鉴定结果如图9所示(引物：EGE-ZXH-007-5’MSD-F/3’MSD-R)。
结论：通过PCR产物测序，表明EH7-5，EH7-10，EH7-15，EH7-19，EH7-22和EH7-27与WT小鼠交配得到F1代。
7.F1代小鼠基因型鉴定
选择F0代鼠尾基因型鉴定结果中阳性小鼠与野生型交配获得具有稳定基因型的F1代小鼠，交配结果如下表5所示。
表5交配结果
F1代基因型鉴定具体如下。
引物设计如F0代小鼠基因型检测的表4所示。
鉴定结果如图10和图11所示。
表6结果表一
| 小鼠ID | 父本ID | 母本ID | DOB | 性别 | 基因型 |
| 1EH7-5 | EH7-10 | C57BL/6 | 2016/5/26 | ♂ | △1483/+ |
| 1EH7-10 | EH7-10 | C57BL/6 | 2016/5/26 | ♂ | △1483/+ |
| 1EH7-30 | EH7-19 | C57BL/6 | 2016/5/26 | ♂ | In1△1459/+ |
| 1EH7-32 | EH7-27 | C57BL/6 | 2016/5/26 | ♂ | In7△1613/+ |
| 1EH7-33 | EH7-27 | C57BL/6 | 2016/5/26 | ♀ | In7△1613/+ |
| 1EH7-35 | EH7-27 | C57BL/6 | 2016/5/26 | ♀ | In7△1613/+ |
| 1EH7-39 | EH7-27 | C57BL/6 | 2016/5/26 | ♀ | In7△1613/+ |
表7结果表二
| 小鼠ID | 父本ID | 母本ID | DOB | 性别 | 基因型 |
| 1EH7-41 | EH7-27 | C57BL/6 | 2016/5/26 | ♀ | In7△1613/+ |
| 1EH7-42 | EH7-27 | C57BL/6 | 2016/5/26 | ♀ | In7△1613/+ |
| 1EH7-44 | EH7-27 | C57BL/6 | 2016/5/26 | ♀ | In7△1613/+ |
| 1EH7-52 | EH7-19 | C57BL/6 | 2016/6/1 | ♀ | In1△1459/+ |
| 1EH7-59 | EH7-19 | C57BL/6 | 2016/6/1 | ♂ | In1△1459/+ |
| 1EH7-60 | EH7-19 | C57BL/6 | 2016/6/1 | ♂ | In1△1459/+ |
| 1EH7-61 | EH7-19 | C57BL/6 | 2016/6/1 | ♂ | In1△1459/+ |
结论：通过PCR鉴定，表明1EH7-5，1EH7-10，1EH7-30，1EH7-32，1EH7-33，1EH7-35，1EH7-39，1EH7-41，1EH7-42，1EH7-44，1EH7-52，1EH7-59，1EH7-60和1EH7-61为阳性杂合子小鼠。
8.Nogo-B敲除小鼠肝脏Western Blot检测
8.1样本信息
选用经过2次剪尾基因型鉴定的Nogo-B敲除小鼠(335，769，ZEH7-8)和Nogo-B野生型小鼠(728)肝脏进行Western Blot检测。
8.2材料与方法
(1)采用的试剂如下表8所示。
表8采用的主要试剂
(2)采用的仪器如下表9所示。
表9主要仪器列表
| 仪器名称 | 厂家 |
| High speed refrigerated centrifuge | XIANGYI |
| VE180 Mini-protean 3 Dodeca | Thermo |
| VE186 TBC | Tanon |
| PowerPac HC Power Supply | BioRad |
| PH meter | Sartorius |
| shaker | Kylin-Bell |
| homogenizer | Fluka |
(3)溶液的配制
1)1.0mol/L Tris·HCl的配制
表10 1.0mol/L Tris·HCl的配制
2)1.74mg/ml(10mmol/L)PMSF的配制
表11 1.74mg/ml(10mmol/L)PMSF的配制
3)10％SDS的配制
表12 10％SDS的配制
4)10％过硫酸胺(APS)的配制
表13 10％过硫酸胺(APS)的配制
5)1.5mol/L Tris·HCl(pH8.8)的配制
表14 1.5mol/L Tris·HCl(pH8.8)的配制
6)0.5mol/L Tris·HCl(pH6.8)的配制
表15 0.5mol/L Tris·HCl(pH6.8)的配制
7)30％Acr/Bic的配制
表16 30％Acr/Bic的配制
8)还原型5XSDS上样缓冲液的配制
表17还原型5XSDS上样缓冲液的配制
9)电泳液缓冲液的配制
表18电泳液缓冲液的配制
10)转移缓冲液的配制
表19转移缓冲液的配制
11)10X丽春红染液的配制
表20 10X丽春红染液的配制
12)TBS缓冲液、TBST缓冲液的配制
表21TBS缓冲液、TBST缓冲液的配制
13)封闭液的配制
表22封闭液的配制
| 脱脂奶粉/BSA | 5g/3g |
| TBST | 100ml |
(4)SDS-PAGE胶的配制
SDS-PAGE分离胶和SDS-PAGE浓缩胶(5％Acrylamide)的配制采用常规方法。
8.3实验方法
(1)蛋白抽提
预冷RIPA蛋白抽提试剂，加入蛋白酶抑制剂Protease inhibitor cocktail(Roche)(磷酸化蛋白需要同时加入磷酸酶抑制剂)。将组织按照10％匀浆(例如10mg加入100ul RIPA裂解液)，进行匀浆。冰上孵育20min后，13000rpm(4℃)离心20min。取上清，分装-80度保存，待测。
(2)BCA法蛋白定量
按照BCA蛋白定量试剂盒使用说明操作，测定蛋白浓度。
1)根据样品数量，试剂A：B＝50：1配制适量BCA工作液，充分混匀。BCA工作液室温24小时内稳定。
2)完全溶解蛋白标准品，取10μl稀释至100μl，使终浓度为0.5mg/ml。
3)将标准品按0，1，2，4，8，12，16，20μl加至96孔板的标准品孔中，并用标准品稀释液补足20μl。
4)将待测样品滴加至样品孔中，用标准品稀释液补足20μl。
5)各孔加入200μl BCA工作液，37℃放置30min。
6)测定A570nm，绘制标准曲线图并计算蛋白浓度。
(3)蛋白浓度调整
以RIPA调整蛋白浓度，样品终浓度为4μg/μl，加入5×蛋白样品缓冲液，95度5min，具体见表23所示。
表23蛋白浓度调整表
| 样本编号 | 样本浓度μg/μl | 制备4ug/ul 800ug总蛋白 | 补充RIPA裂解液 |
| ZEH7-8 | 22.7 | 35 | 165 |
| 335 | 15.1 | 53 | 147 |
| 728 | 19.4 | 41 | 159 |
| 769 | 16.4 | 49 | 151 |
(4)Western Blot实验
1)根据目的蛋白的分子量，配制10％梯度胶，浓缩胶浓度为5％。
2)待检测蛋白样品上样量：40μg/孔。
3)电泳条件：浓缩胶恒压90V，约30min；分离胶恒压120V，通过预染蛋白marker来确定电泳停止时间。
4)湿转法，转膜条件：300mA恒流；0.45μm孔径PVDF膜，转膜时间90min。转膜完成后丽春红染色试剂对膜进行染色，观察转膜效果。
5)封闭：将膜完全浸没于5％BSA-TBST中，水平摇床孵育2h(RT)。
6)一抗孵育：5％BSA-TBST稀释一抗，4℃水平摇床孵育过夜。
表24不同的一抗及其稀释表
| 膜编号 | 一抗名称 | 来源 | 稀释比例 | 稀释后体积 |
| 1 | Nogo+B2 | Rabbit | 1:1000 | 4ml |
| 2 | GAPDH | Mouse | 1:1000 | 4ml |
7)次日，洗膜：TBST洗3次，每次10min。
8)二抗孵育：5％BSA-TBST稀释二抗：山羊抗兔，山羊抗鼠IgG(H+L)HRP 1:10000，室温孵育40min。洗膜：TBST洗膜3次，每次10min。
9)ECL滴加到膜的蛋白面，反应3min；胶片曝光：10s-5min(曝光时间随不同光强度而调整)，显影2min，定影。
8.4 Western Blot检测结果
检测得到的结果如图12所示。其中，728为Nogo-B野生型小鼠(+/+)，335，769，ZEH7-8为Nogo-B敲除小鼠(-/-)。
需要注意的是，除非另有说明，本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。除非另外具体说明，否则在这些实施例中阐述的部件和步骤的相对步骤、数字表达式和数值并不限制本发明的范围。在这里示出和描述的所有示例中，除非另有规定，任何具体值应被解释为仅仅是示例性的，而不是作为限制，因此，示例性实施例的其他示例可以具有不同的值。
在本发明的描述中，需要理解的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本发明的描述中，“多个”的含义是两个以上，除非另有明确具体的限定。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各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，或者对其中部分或者全部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范围，其均应涵盖在本发明的保护范围当中。
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Claims (8)
1.一种基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法，其特征在于，包括步骤：
设计高效的识别特定基因EGE-ZXH-007的sgRNA；
将所述sgRNA连入质粒载体上，然后进行体外转录，得到可进行显微注射的Cas9/sgRNA；
将所述可进行显微注射的Cas9/sgRNA显微注射到小鼠受精卵中，然后对出生的小鼠进行基因型检测和鉴定，确认最终得到Nogo-B敲除模式小鼠。
2.根据权利要求1所述的基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法，其特征在于：
所述sgRNA的序列如SEQ ID NO.1和SEQ ID NO.2所示。
3.根据权利要求1所述的基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法，其特征在于：
所述质粒载体为带T7启动子的质粒载体。
4.根据权利要求1所述的基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法，其特征在于：
在将所述sgRNA连入质粒载体上之前，还包括Cas9/sgRNA构建和活性检测的步骤。
5.根据权利要求4所述的基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法，其特征在于：
所述Cas9/sgRNA构建和活性检测具体包括：将所述sgRNA连入pCS载体，得到连接产物Cas9/sgRNA；将所述Cas9/sgRNA转化，然后测序验证正确；再对Cas9/sgRNA进行活性检测。
6.权利要求1-5任一项所述的方法获得的Nogo-B敲除模式小鼠在Nogo-B的功能研究中的应用。
7.一种获得Nogo-B敲除模式小鼠的试剂盒，其特征在于：
所述试剂盒包括能识别特定基因EGE-ZXH-007的sgRNA。
8.根据权利要求7所述的获得Nogo-B敲除模式小鼠的试剂盒，其特征在于：
所述sgRNA的序列如SEQ ID NO.1和SEQ ID NO.2所示。
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