CN106191057A - 一种用于敲除人CYP2E1基因的sgRNA序列、CYP2E1基因缺失细胞株的构建方法及其应用 - Google Patents
一种用于敲除人CYP2E1基因的sgRNA序列、CYP2E1基因缺失细胞株的构建方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种用于敲除人CYP2E1基因的sgRNA序列，所述sgRNA的靶DNA序列为SEQ ID NO:1、SEQ ID NO:2所示序列中的至少一条。本发明还提供了一种敲除人胚胎肾脏细胞CYP2E1基因的方法，为利用CRISPR/Cas系统在人胚胎肾脏细胞中对CYP2E1基因进行改造。本发明还提供了一种CYP2E1基因敲除细胞株，CYP2E1参与机体重要的代谢功能，本发明提供的CYP2E1基因敲除细胞株为外源性化学物或者外源性毒物在体内的代谢研究提供了有效的平台，对于慢性疾病(如酒精性肝脏疾病及糖尿病)及肿瘤相关疾病的研究提供了有力工具。
Description
技术领域
本发明涉及基因工程领域，具体涉及一种用于敲除人CYP2E1基因的gRNA序列、CYP2E1基因缺失细胞株的构建方法及其应用。
背景技术
CYP2E1是细胞色素P450家族里非常重要的成员之一，CYP2E1基因位于第10号染色体上，有11413bp，含有9个外显子和8个内含子，编码含有493个氨基酸的蛋白。CYP2E1主要存在于肝脏和肾脏细胞的内质网和线粒体内，主要参与外来化学物的体内代谢和生物转化，还参与了机体的氧化应激、脂质过氧化、细胞凋亡与自噬、炎症反应等过程，会对机体造成损伤及产生毒性。因此，急需要构建一种CYP2E1基因缺陷型人胚胎肾脏细胞株，用于CYP2E1相关的药物和毒物代谢研究、外源性化学物毒性研究、致癌性研究以及药物-药物交互作用研究，为进一步进行肿瘤相关药物研究和外源化学物代谢研究提供良好的工具和平台。
CRISPR/Cas9系统是一种后天免疫防御系统，用以保护细菌或古细菌免受外来质粒或噬菌体的侵入，这类细菌或古细菌基因组的CRISPR序列能表达与入侵者基因组序列相识别的RNA，在CRISPR相关酶(CAS9)的作用下切割外源基因组DNA，达到抵制入侵的目的，经过人为改造后，CRISPR/Cas9系统可以实现在真核细胞中高度灵活且特异的基因组编辑，是目前基因组编辑领域最受欢迎的新一代基因组编辑技术，目前该技术已被用于构建各类基因敲除细胞系和基因敲除动物模型。
现有实验技术中，与CRISPR/Cas9系统最为相似的是siRNA靶向的基因沉默技术，siRNA靶向的基因沉默是在转录或者转录后水平实现基因沉默，其对基因表达的沉默作用往往并不彻底，达不到预期的沉默效果。现有报道的siRNA(例：siCYP2E1)在mRNA或蛋白水平的沉默不彻底，无法完全沉默基因的表达，不能构建真正的CYP2E1基因缺陷细胞株。
目前，有必要提供一种不仅能实现沉默彻底、且能长期稳定体外培养的CYP2E1基因缺陷型人胚胎肾脏细胞株。
发明内容
为解决上述问题，本发明提供了一种用于敲除人CYP2E1基因的sgRNA序列、CYP2E1基因缺失细胞株的构建方法及其应用。
第一方面，本发明提供了一种用于敲除人CYP2E1基因的sgRNA序列，所述sgRNA的靶DNA序列为SEQ ID NO:1、SEQ ID NO:2所示序列中的至少一条。
第二方面，本发明提供了一种敲除人胚胎肾脏细胞CYP2E1基因的方法，为利用CRISPR/Cas系统在人胚胎肾脏细胞中对CYP2E1基因进行改造，具体包括如下步骤：
(1)人工合成如第一方面所述靶DNA序列及其互补链；
(2)将所合成的核酸片段插入到sgRNA骨架表达质粒载体的多克隆位点并转化，挑单克隆菌株，提取sgRNA重组质粒，测序鉴定获得测序正确的sgRNA重组质粒；其中，sgRNA骨架表达质粒载体还表达Cas9核酸酶；
(3)将sgRNA重组质粒转染人胚胎肾脏细胞，即得敲除CYP2E1基因的人胚胎肾脏细胞。
优选地，所述步骤(2)具体包括：将所合成的SEQ ID NO:1及SEQ ID NO:2所示序列的核酸对分别插入到sgRNA骨架表达质粒载体的多克隆位点并转化，挑单克隆菌株，提取sgRNA重组质粒，测序鉴定获得测序正确的sgRNA重组质粒；其中，sgRNA骨架表达质粒载体还表达Cas9核酸酶；
所述步骤(3)具体包括：将步骤(2)所得的两种sgRNA重组质粒共转染人胚胎肾脏细胞，即得敲除CYP2E1基因的人胚胎肾脏细胞。
第三方面，本发明提供了一种CYP2E1基因缺失细胞株的构建方法，为采用有限稀释法对第二方面所得的敲除CYP2E1基因的人胚胎肾脏细胞进行传代筛选，获得稳定敲除CYP2E1的人胚胎肾脏细胞。
第四方面，本发明提供了一种CYP2E1基因缺失细胞株，为如采用第三方面所述的CYP2E1基因缺失细胞株的构建方法所制得。
第五方面，本发明提供了一种如第一方面所述的用于敲除人CYP2E1基因的sgRNA序列在敲除CYP2E1基因中的应用。
第六方面，一种用于在人基因组中进行CYP2E基因定点敲入试剂盒，包括如下(1)-(3)中的任一种：
(1)如第一方面所述的用于敲除人CYP2E1基因的sgRNA序列；
(2)如第二方面所述的sgRNA重组质粒；
(3)如第四方面所述的CYP2E1基因缺失细胞株。
本发明提供了的技术方案具有如下有益效果：
本发明提供的技术方案利用CRISPR/Cas9技术对CYP2E1基因敲除，实现了在基因组水平对基因的沉默作用，有效改进了siRNA(例：siCYP2E1)在mRNA或蛋白水平的沉默不彻底或者无法沉默基因表达的缺点。
附图说明
图1为本发明实施例提供的SgRNA重组质粒构建模式图；
图2为本发明实施例提供的倒置荧光显微镜观察细胞荧光结果；
图3为本发明实施例提供的SURVEYOR核酸酶消化后PCR片段结果；
图4为本发明实施例提供的293FT-ko-45#、293FT-ko-46#在靶点位置的缺失突变序列示意；
图5为本发明实施例提供的CYP2E1-Knockout细胞株mRNA表达量检测结果；
图6为本发明实施例提供的CYP2E1-Knockout细胞株Protein表达量检测结果。
具体实施方式
以下所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。
本发明实施例中无特别说明外，所用试剂及耗材均为市售商品。
本发明技术方案可通过如下实施例实现：
(1)sgRNA设计：
分别针对CYP2E1的第二、第三和第七外显子(Exon2,Exon3,Exon7)设计sgRNA序列。
设计、合成的三组分别针对CYP2E1第二、第三和第七外显子的sgRNA序列具体分组与命名见表1：
表1CYP2E1 sgRNA oligo序列(Tab.1 The sequences of CYP2E1 sgRNA oligo)
分别在sgRNA两端加酶切位点，在每条sgRNA序列的正义链的5’端添加CACC，反义链的5’端添加AAAC，从而形成与PX461质粒经Fast Digest Bbs I酶切后互补的粘性末端。如果正义链的5’端第一个碱基不是G，则在5’端CACC后面增加一个G，相应的反义链3’端再增加一个C。设计完成后的sgRNA送上海杰瑞公司进行引物合成。(画横线为sgRNA)
本发明SEQ ID NO:1、SEQ ID NO:2所示序列分别对应表格1中SEQ ID NO:8、SEQID NO:9画横线部分，具体地，SEQ ID NO:1、SEQ ID NO:2所示序列分别为GGAAGGACATCCGGCGGTTT和ACCCTCCGGAACTATGGGAT。
重组质粒的构建与鉴定，构建流程模式如图1所示
①PX461为含有U6启动子的sgRNA骨架表达载体，表达具有Cas9 D10A切口酶突变的Cas9n，带有GFP绿色荧光蛋白基因和氨苄青霉素抗性。用Fast Digest Bbs I对PX461进行酶切，DNA凝胶电泳后回收线性化的载体。
②用T4 PNK分别对表1中的三组sgRNA oligo序列进行磷酸化和退火；用T4ligase将线性的PX461质粒载体分别与退火后的三组sgRNA双链序列室温连接1h。连接产物转化感受态细菌Trans 109，冰浴30min,42℃45s，冰上2min。在氨苄抗性的LB平板上筛选克隆。挑取阳性克隆摇菌，送测序。测序引物为U6启动子的正向引物序列，5’-GAGGGCCTATTTCCCATGATTCC-3’(SEQ ID NO:15)。测序正确的克隆提取重组质粒。
③所得重组质粒共有三组(6种)，针对第二外显子的一组质粒命名为PX461-E2-1和PX461-E2-2(分别对应Exon2的SgRNA-E2-1、SgRNA-E2-2构建的质粒)，针对第三外显子的一组质粒命名为PX461-E3-1和PX461-E3-2(分别对应Exon3的SgRNA-E3-1、SgRNA-E3-2构建的质粒)，针对第七外显子的一组质粒命名为PX461-E7-1和PX461-E7-2(分别对应Exon7的SgRNA-E7-1、SgRNA-E7-2构建的质粒)。
(2)细胞培养和细胞转染
①293FT细胞培养条件：DMEM培养基(含10％胎牛血清)、5％CO2、37℃恒温培养。
转染前24h，将293FT细胞以5×105/孔接种至6孔板中培养，转染时细胞融合度达到60％-70％。使用lipo2000转染试剂分别将上述每组(Exon2、3、7)对应的2种质粒同时转染一孔293FT细胞，等量的PX461质粒作为阴性对照，6孔板质粒转染量一般为2ug/孔,质粒预转染试剂之比为1:2-2.5。
②转染后24h观察转染效率。利用倒置荧光显微镜观察荧光细胞百分比，以确定转染效率，结果如图2所示。
(4)提取细胞基因组DNA
细胞转染后48h，将293FT细胞进行消化，取一部分(一般为消化后细胞重悬液体积1/3)进行传代保种。一部分(一般为消化后细胞重悬液体积2/3)用GeneJETTMGenomic DNAPurification Kit提取基因组DNA。
①将细胞收集于离心管，每管5X106个细胞，用移液器缓慢吹打，250g离心5min，弃上清，加PBS重悬细胞，再次重复离心，已去除细胞中残留培养基。
②用200ul PBS重悬细胞，每管加入200ul裂解液和20ul蛋白酶K,充分震荡、混合均匀。
③56℃摇床孵育10min，期间每3-4分钟震荡混匀一次，以保证细胞裂解充分。
④加入20ul RNAase A，震荡混匀，室温孵育10min。
⑤加入400ul 50％ethanol，用枪震荡混匀或者震荡混匀。
⑥将上述MiX加入试剂盒提供的Column柱中，6000g离心，1min，将DNA收集柱转移至新的2ml收集管中。
⑦加入500ul wash bufferⅠ，8000g离心1min，弃废液。再加入500ul wash bufferⅡ,12000g,离心3min。
⑧加入200ul Elution Buffer至收集柱滤膜中央，室温孵育2min，8000g离心，1min，即可得所需DNA样品。
(5)PCR反应条件和SURVEYOR分析检测
①SURVEYOR PCR反应：只有能被sgRNA识别，Cas9切割的DNA序列存在，SURVEYOR结果呈现阳性(3条带)，因此需要首先针对CYP2E1基因上三个不同的外显子进行分析检测，分别设计3对跨Cas9蛋白切割位点的SURVEYOR PCR引物对，并进行引物特异性检测,引物序列见表2。
表2SURVEYOR PCR反应引物序列
Tab.2 PCR primer
②用Phusion超保真DNA聚合酶进行PCR扩增，参照说明书50ul体系，基因组DNA100ng，50ul反应体系如下表所示。
| 组分 | 数量(uL) |
| H2O | to 50 |
| Phusion HF buffer,5X | 10 |
| dNTPs,2.5mM | 4 |
| Phusion polymerase | 0.5 |
| Forward primer | 2.5 |
| Reverse primer | 2.5 |
| template DNA | 100ng |
| Total | 50 |
程序如下：
取反应后PCR产物5ul进行琼脂糖凝胶电泳检测其特异性。
③SURVEYOR分析步骤如下：
③.1用QIAquick PCR purification Kit试剂盒进行PCR产物纯化，将回收产物稀释至40ng/ul，按照SURVEYOR分析试剂盒说明书步骤进行检测.
③.2DNA杂化双链形成(退火反应)体系：
| 组分 | 数量(μl) |
| Taq PCR buffer,10× | 2 |
| Normalized PCR product,20ngμl-1 | 18 |
| Total volume | 20 |
反应条件：
| 循环次数 | 条件 |
| 1 | 95℃,10min |
| 2 | 95-85℃,-2℃s-1 |
| 3 | 85℃,1min |
| 4 | 85-75℃,-0.3℃s-1 |
| 5 | 75℃,1min |
| 6 | 75-65℃,-0.3℃s-1 |
| 7 | 65℃,1min |
| 8 | 65-55℃,-0.3℃s-1 |
| 9 | 55℃,1min |
| 10 | 55-45℃,-0.3℃s-1 |
| 11 | 45℃,1min |
| 12 | 45-35℃,-0.3℃s-1 |
| 13 | 35℃,1min |
| 14 | 35-25℃,-0.3℃s-1 |
| 15 | 25℃,1min |
| 16 | 25-4℃,-0.3℃s-1 |
| 17 | 4℃,hold |
③.3SURVEYOR核酸酶消化(在冰上操作)：
反应体系：
| 组分 | 用量(μl) | 终浓度 |
| Annealed heteroduplex | 20 | |
| MgCl2stock solution supplied with kit,0.15M | 2.5 | 15mM |
| ddH2O | 0.5 | |
| SURVEYOR nuclease S | 1 | 1× |
| SURVEYOR enhancer S | 1 | 1× |
| Total | 25 |
反应条件：充分震荡、混匀上述mixture,42℃30min.
③.4取10ul样品，用2％琼脂糖凝胶进行分析。用凝胶定量软件计算切割效率，公式为 fcut＝(b+c)/(a+b+c)，其中Indel为缺失比率，fcut为切割比率，a为未被切割条带的灰度值，b和c表示切割产生的新条带的灰度值。
选择Indel(％)最高的那一组转染的293FT细胞做下一步的接种和筛选。
SURVEYOR核酸酶消化部分结果如图3所示，实验结果表明仅有针对Exon3设计的sgRNA构建的PX461-E3-1和PX461-E3-2质粒组有效，SURVEYOR结果为阳性。
(6)筛选稳定敲除CYP2E1的293FT细胞株
为进一步获得稳定敲除CYP2E1的293FT细胞株，我们采用有限稀释法，将传代保种的经过转染的293FT单细胞接种至96孔板中。
操作步骤如下：
①用胰蛋白酶消化细胞并计数，采用有限稀释法稀释至100ul培养基0.5个细胞，按每孔100ul细胞稀释液加至96孔板中。
②接种后第5至7天用显微镜观察细胞生长状况并初步筛选出单克隆细胞，待细胞长满96孔板底时，再用胰蛋白酶消化细胞并转移至24孔板中。
③待细胞长满24孔板底时，一部分细胞用于传代留种，一部分细胞提取其基因组DNA，经PCR扩增后测序，测序结果与原基因组进行比对，检测靶向敲除CYP2E1基因是否成功。
测序结果发现，293FT-ko-45#在靶点位置造成了35bp的缺失突变，293FT-ko-46#在靶点位置也造成了37bp的缺失突变，如图4所示。
④挑选经测序验证的成功敲除CYP2E1基因的单克隆细胞，培养扩增至6孔板，一部分细胞用于传代留种，一部分细胞用M-PER Mammalian Protein Extraction Reagent提取蛋白，用于Western blot检测CYP2E1的蛋白表达水平，另一部分细胞用Trizol法提取RNA，用于RT-qPCR检测CYP2E1的mRNA表达水平。
结果如图5、6所示。图5为CYP2E1-Knockout细胞株mRNA表达量检测结果；图6为CYP2E1-Knockout细胞株Protein表达量检测结果。
相对于传统的基因敲除方法不仅流程繁琐，对技术要求高，而且费用昂贵，成功率相对较低。本发明采用的CRISPR-Cas9技术是第四代基因编辑方法，其易于操作，效率更高，费用低廉。本发明利用CRISPR-Cas9技术构建的CYP2E1基因敲除细胞模型为CY2E1代谢相关的研究提供了有效平台。具体有益效果如下：
(1)利用CRISPR/Cas9技术对CYP2E1基因敲除，实现了在基因组水平对基因的沉默作用，有效改进了siRNA(例：siCYP2E1)在mRNA或蛋白水平的沉默不彻底或者无法沉默基因表达的缺点。
(2)CYP2E1参与机体重要的代谢功能，CYP2E1基因敲出细胞株的建立为外源性化学物或者外源性毒物在体内的代谢研究提供了有效的平台。
(3)CYP2E1基因敲除细胞株对于慢性疾病(如酒精性肝脏疾病及糖尿病)及肿瘤相关疾病的研究提供了有力工具。
(4)CYP2E1基因敲除细胞株可用于CYP2E1代谢相关外源性化学物毒性、致癌性的研究以及药物之间的交互作用研究。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (6)
1.一种用于敲除人CYP2E1基因的sgRNA序列，其特征在于，所述sgRNA的靶DNA序列为SEQ ID NO:1、SEQ ID NO:2所示序列中的至少一条。
2.一种敲除人胚胎肾脏细胞CYP2E1基因的方法，其特征在于，为利用CRISPR/Cas系统在人胚胎肾脏细胞中对CYP2E1基因进行改造，具体包括如下步骤：
(1)人工合成如权利要求1所述的靶DNA序列及其互补链；
(2)将所合成的核酸片段插入到sgRNA骨架表达质粒载体的多克隆位点并转化，挑单克隆菌株，提取sgRNA重组质粒，测序鉴定获得测序正确的sgRNA重组质粒；其中，sgRNA骨架表达质粒载体还表达Cas9核酸酶；
(3)将sgRNA重组质粒转染人胚胎肾脏细胞，即得敲除CYP2E1基因的人胚胎肾脏细胞。
3.一种CYP2E1基因缺失细胞株的构建方法，其特征在于，为采用有限稀释法对如权利要求2所得的敲除CYP2E1基因的人胚胎肾脏细胞进行传代筛选，获得稳定敲除CYP2E1的人胚胎肾脏细胞。
4.一种CYP2E1基因缺失细胞株，其特征在于，为如采用如权利要求3所述的CYP2E1基因缺失细胞株的构建方法所制得。
5.一种如权利要求1所述的用于敲除人CYP2E1基因的sgRNA序列在敲除CYP2E1基因中的应用。
6.一种用于在人基因组中进行CYP2E基因定点敲入试剂盒，其特征在于，包括如下(1)-(3)中的任一种：
(1)如权利要求1所述的用于敲除人CYP2E1基因的sgRNA序列；
(2)如权利要求2所述的sgRNA重组质粒；
(3)如权利要求4所述的CYP2E1基因缺失细胞株。
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Cited By (27)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107177631A (zh) * | 2017-06-26 | 2017-09-19 | 中国农业大学 | 利用CRISPR‑CAS9技术敲除NRK细胞Slc22a2基因的方法 |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| CN111849987A (zh) * | 2020-07-29 | 2020-10-30 | 华农（肇庆）生物产业技术研究院有限公司 | Viperin功能缺失的细胞系的构建方法及其应用 |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| CN113801852A (zh) * | 2021-10-18 | 2021-12-17 | 齐齐哈尔医学院 | 一种gpd1l缺失人胚胎干细胞细胞株及其构建方法和应用 |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| US12031126B2 (en) | 2023-12-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703507A (zh) * | 2012-05-18 | 2012-10-03 | 深圳市疾病预防控制中心 | 特异抑制肝细胞CYP2E1基因表达的shRNA慢病毒表达载体及其构建方法与应用 |
| EP3009511A2 (en) * | 2015-06-18 | 2016-04-20 | The Broad Institute, Inc. | Novel crispr enzymes and systems |
| CN105624191A (zh) * | 2015-12-24 | 2016-06-01 | 江苏大学 | 一种建立cyp2d1基因敲除大鼠模型的方法 |
-
2016
- 2016-07-06 CN CN201610528473.8A patent/CN106191057B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703507A (zh) * | 2012-05-18 | 2012-10-03 | 深圳市疾病预防控制中心 | 特异抑制肝细胞CYP2E1基因表达的shRNA慢病毒表达载体及其构建方法与应用 |
| EP3009511A2 (en) * | 2015-06-18 | 2016-04-20 | The Broad Institute, Inc. | Novel crispr enzymes and systems |
| CN105624191A (zh) * | 2015-12-24 | 2016-06-01 | 江苏大学 | 一种建立cyp2d1基因敲除大鼠模型的方法 |
Non-Patent Citations (4)
| Title |
|---|
| WANG, X ET AL.: "Characterization of novel cytochrome P450 2E1 knockout rat model generated by CRISPR/Cas9", 《BIOCHEMICAL PHARMACOLOGY》 * |
| ZONG, HH ET AL.: "Cytochrome P-450 CYP2E1 knockout mice are protected against high-fat diet-induced obesity and insulin resistance", 《AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM》 * |
| 姚聪 等: "CYP1B1基因敲除对高脂膳食诱导小鼠肥胖的抑制作用", 《环境与健康杂志》 * |
| 韩露 等: "CYP2E1在酒精性肝病中的作用", 《安徽医药》 * |
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