血管动力尾静脉注射对小鼠肝细胞基因的本构和诱导系统的修饰
Summary
水动力尾静脉注射转的融合载体, 可以稳定转染小鼠肝细胞在体内.在这里, 我们提出了一个实用的转染系统的协议, 使一个单一的转基因或联合本构和强力霉素诱导表达的转基因或和平 shRNA 在肝脏的长期本构表达。
Abstract
在肝癌的研究模式, 再生, 炎症和纤维化, 灵活的系统为体内基因表达和沉默是非常有用的。以转为基础的水动力尾静脉注射是一种有效的成年小鼠肝细胞遗传操作方法。除了本构式转基因表达外, 该系统还可用于更先进的应用, 如 shRNA 介导的基因敲除, CRISPR/Cas9 系统诱导基因突变或诱导系统的暗示。将本构 CreER 表达与转基因或 shRNA 选择的诱导表达结合起来, 作为这一技术的一个例子。我们涵盖了多步骤的过程, 从准备的睡美人-转结构, 注射和治疗小鼠, 并准备肝组织进行分析的免疫。该系统是实现肝细胞复杂遗传操作的可靠而有效的方法。它是特别有用的结合与 loxP 的小鼠品系, 可用于各种模型的研究肝病。
Introduction
慢性肝病是一个主要的健康负担全球1。动物研究模型是肝脏疾病研究的重要工具, 有助于解答肝脏再生、肝脏炎症、脂肪以及肝癌2等复杂问题。大量的这些动物模型依赖于肝细胞的基因修饰。因此, 有效的工具, 操纵基因表达在肝细胞是有益的3。建立的方法, 如育种的基因工程小鼠菌株或病毒载体的生成肝细胞感染要么费时, 港口安全关注, 或屈服于肝细胞的不良转基因表达在体内4,5. 水动力尾静脉注射 (HTVI) 是一种替代方法的体内转染肝细胞允许方便, 快速, 和经济高效的基因功能的审讯。对于 HTVI, 携带所需 DNA 序列的载体被溶解在相当于注射动物体重10% 的盐水量中。然后在5-10 秒6内将该解决方案注入尾部静脉。超过心脏输出, 盐水从下腔静脉流入肝静脉, 导致肝脏扩张和肝细胞的水动力转染7。为了实现稳定的基因组集成, 该方法已与基于转的矢量 (如休眠美人-转系统) 相结合。该系统介导的重组的目标载体与基因组重组网站的催化睡美人-转8,9。对于肝纤维化或癌变的模型, 在疾病模型的某些时间点过度或沉默基因往往是可取的。为此, 可诱导基因表达的工具, 如 LoxP 系统或四环素诱导基因表达系统 (春节) 可以使用10。
在这里, 我们描述了一个协议的在体内转染小鼠肝细胞使用 HTVI 的睡美人转的系统。除了一个在肝脏特异启动子控制下的转基因的稳定、本构表达的协议外, 我们还描述了一种更先进的向量系统, 它将本构型他莫昔芬重组 (CreER) 表达与可诱导表达的转基因或 rna 适应的 shRNA (和平 shRNA), 称为 pTC 的春节系统11。在这个向量系统中, 可诱导的转基因或和平 shRNAs 为四环素相关的表达被克隆到骨干载体与重组克隆系统, 允许快速和容易生成新的载体12。本视频指南包括制备合适的载体, 注射和治疗小鼠, 以实现诱导转基因/和平 shRNA 表达, 最后准备肝脏组织进行分析。本协议中描述的方法旨在使任何选择的基因 loxP 介导的小鼠系统的表达或击倒, 使得它成为一种广泛适用的肝病研究系统。
Protocol
所有动物实验都是根据实验室动物的护理和使用指南进行的, 并得到主管当局的批准 (Regierung Oberbayern、慕尼黑、德国和斯坦福机构动物保育和使用委员会,斯坦福, 加利福尼亚, 美国)。在补充表 S1 中提供了所有用于克隆的质粒的清单 (步骤1至 4)。 1. 本构基因表达的转基因克隆 设计引物为转基因放大13,14。 将总署 (TTAATT…
Representative Results
水动力尾静脉注射转染效果:单次注射转染水力的小鼠肝细胞百分比是可变的, 取决于注射量、注射时间、注入 DNA 量和注入结构的大小等多个参数6, 22,23。此外, 转染效率一般较低, 在较大的动物, 较大的血管直径, 以及较大的正弦面积导致整体压力下降。为了直观地显示转染效率, CreER 转结?…
Discussion
水动力尾静脉注入肝细胞的转染已成为15年前6以来建立的一种方法。注入的体积超过心脏输出, 从下腔静脉流向肝脏的窦7, 导致转染约 10-20%, 在某些情况下多达40% 的肝细胞25,26。成功转染的预测因子是每次注入时间的注入量22,23。因此, 低转染效率 (图 1A, 左面板…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到德国德意志 Krebshilfe (111289 号至 ue)、露西尔的儿童健康基金会的支持 (欧内斯特和阿米莉亚-奖授予博士后奖学金-UL1 RR025744 至 ue)。我们感谢马克. 凯博士的向量结构和实验建议和朱利安博士鼠和实验支持。
Materials
| General Material | |||
| GeneRuler 1 kb Plus DNA Ladder | Thermo Fisher | #SM1331 | DNA ladder for electrophoresis |
| Tissue-Tek O.C.T. | Sakura | 4583 | embedding of cryo-sections |
| Biozym LE Agarose | Biozym | 840004 | |
| Ethidium bromide | Sigma-Aldrich | E7637-1G | |
| D(+)-Saccharose | Carl Roth | 4621.1 | For sweetening of the doxycyline solution |
| Ampicillin Sodium Salt | AppliChem | A0839,0010 | For selection of Amp-resistant clones |
| LB Agar (Luria/Miller) | Carl Roth | X969.1 | |
| LB Broth (Luria/Miller) | Carl Roth | X968.1 | |
| S.O.C. Medium | Thermo Fischer | 15544034 | |
| Gentamicin sulfate | AppliChem | A1492,0001 | For selection of Gentamicin-resistant clones |
| Roti-Histofix 4 % | Fa. Roth | P087.6 | para-formaldehyde solution |
| T4 DNA Ligase | New England BioLabs | M0202S | |
| GatewayTM LR ClonaseTM II Enzyme Mix | invitrogen/ThermoFisher | 11791-020 | contains LR-clonase enzyme mix II and proteinase K |
| DB3.1 Competent Cells | Thermo Fisher | 11782-018 | |
| Stbl3 Chemically Competent E. coli | Thermo Fisher | C737303 | |
| Name | Company | Catalog Number | Comments |
| Restriction Enzymes | |||
| PacI | New England BioLabs | R0547S | |
| AscI | New England BioLabs | R0558S | |
| FseI | New England BioLabs | R0588S | |
| SacI | New England BioLabs | R0156S | |
| SpeI | New England BioLabs | R0133S | |
| KpnI | New England BioLabs | R0142S | |
| NotI | New England BioLabs | R0189S | |
| XhoI | New England BioLabs | R0146S | |
| BfuAI | New England BioLabs | R0701S | |
| Name | Company | Catalog Number | Comments |
| Kits | |||
| QIAquick Gel Extraction Kit | Qiagen | 28704 | For DNA Extraction from gel |
| NucleoSpin Gel and PCR Clean Up | Macherey & Nagel | 740609.10 | |
| NucleoBond PC20 | Macherey & Nagel | 740571 | Plasmid extraction (Mini prep) |
| NucleoBond PC500 | Macherey & Nagel | 740574 | Plasmid extraction (Maxi prep) |
| Phusion High-Fidelity DNA Polymerase | Thermo Fisher | F530S | |
| Name | Company | Catalog Number | Comments |
| Materials for Mouse Experiments | |||
| Injekt Syringe F 1 ml | Braun | 9166017V | For intraperitoneal injection |
| Omnifix Luer 3 ml | Braun | 4616025V | For intravenous injection |
| Sterican Cannula 24G | Braun | 4657675 | |
| Sterican Cannula 27G | Braun | 4657705 | |
| Tamoxifen | Sigma-Aldrich | T5648-1G | For CreER activation |
| Corn oil | Sigma-Aldrich | C8267-500ML | Carrier for tamoxifen injections |
| Doxycycline hyclate | AppliChem | A2951,0025 | Activation of tetracycline-dependent expression |
| Injekt 10 ml Syringe | Braun | 4606108V | |
| Filtropur S 0.2 | Sarstedt | 831,826,001 | For filtration of doxycycline |
| NaCl 0,9% | Braun | 3200905 | Carrier for intravenous injections |
| Falcon Conical Tube 50ml | Corning Life Science | 352095 | |
| Infrared Lamp | N/A | N/A | For warming of mouse tail |
| IVIS | Perkin Elmer | 124262 | In vivo imaging system |
| Name | Company | Catalog Number | Comments |
| Plasmids for cloning of sleeping beauty-transposon vectors for HTVI. | |||
| pTC | n/a | Vector for constitutive gene expression, ref. 15 | |
| pEN_TTmcs | Addgene #25755 | Entry vector for inducible gene expression, ref. 19 | |
| pEN_TTGmiRc2 | Addgene #25753 | Entry vector for inducible miR-shRNA expression with co-expression of GFP, ref. 19 | |
| pEN_TTmiRc2 | Addgene #25752 | Entry vector for inducible miR-shRNA expression without co-expression of GFP, ref. 19 | |
| pTC ApoE-Tet | Addgene #85578 | Expression vector for inducible gene or miR-shRNA expression with ApoE.HCR.hAAT promotor, ref. 11 | |
| pTC-CMV-Tet | Addgene #85577 | Expression vector for inducible gene or miR-shRNA expression with CMV promotor, ref. 11 |
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Citar este artículo
Hubner, E. K., Lechler, C., Rösner, T. N., Kohnke-Ertel, B., Schmid, R. M., Ehmer, U. Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection. J. Vis. Exp. (132), e56613, doi:10.3791/56613 (2018).