在体内的器官型小鼠视网膜Wholemount文化
Summary
此视频文章演示器官视网膜wholemount文化和cytospin一个外生的诱导效应分析程序的建立。器官型视网膜wholemount文化模仿<em>在体内</em>情况，同时规避经典的小鼠动物模型的缺点，大大促进了实验操作的小鼠视网膜的无障碍。
Abstract
消融基因动物模型分析的目标是招收特定视网膜基因功能的经典战略。然而，转基因，基因敲除小鼠模型的视网膜特定的或有条件的往往显示早期的杀伤力或遭受严重畸形，防止超越胚胎或产后早期阶段的分析。
原代细胞培养是一种替代，以探讨外源重组的因素，在受控环境中的基因或siRNA介导的基因敲除表达的影响。游离细胞培养已减少，到达靶细胞的内源性信号的优势，从而促进确定触发后药理操纵的外生影响。但是，重要的细胞间的相互作用是最初破坏酶消化或机械分离，即使重新聚合 1 retinospheroid文化。
相比之下，器官视网膜wholemount文化提供系统在体内的情况接近的生理与神经元的相互作用和连接仍然保留 2-5 。
在这个视频文章中，我们提供了一步一步的示范（1）建立体内器官包括视网膜清扫胚胎，产后和成年鼠的眼睛（2）进行分析的分离和cytospin神经过程的特殊性wholemount文化例如器官wholemounts视网膜细胞凋亡和细胞增殖，培养后的外源重组的因素存在。
Protocol
所有的设备和试剂必须购买无菌或需要加热或蒸汽消毒或用70％乙醇消毒。 作者状态，对动物的实验是根据欧洲共同体理事会指令（86/609/EEC），美国国立卫生研究院的机构动物所规定的实验程序和法规的动物护理和使用准则保养和使用委员会（IACUC）在杜伊斯堡 – 埃森大学（德国）。 第1部分：不同发育阶段的小鼠的眼睛剜除剜除的胚胎?…
Discussion
利用小鼠器官视网膜wholemount文化2-5以上的解离，单层，retinospheroid或重新聚合的3D球体在保存神经元的相互作用和连接谎言文化，模仿体内的情况。 2前报告相比，我们的视频的文章提供了一个在剜除小鼠的眼睛，包括晶状体和玻璃体切除不损害视网膜的不同发育阶段的视网膜剥离的特殊性详细演示。晶状体和玻璃体切除药理操作至关重要，既阻碍物质的视网膜层的访问。?…
Acknowledgements
作者想感谢最初帮助建立的器官文化和技术援助的美国Laub和美国Gerster E.德拉罗萨和人工智能Valenciano。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Mice | Animal | Charles Rivers Laboratories | ||
| Dissection microscope | Tool | ZEISS | ||
| PBS | Reagent | Sigma | PBS should be cold (> 4°C) and sterile | |
| Dulbecco`s modified eagle`s medium / nutrient mixture F-12 Ham | Reagent | Sigma | D 8900 | DMEM / F-12 |
| Apo-transferin | Reagent | Sigma | T 1147 | |
| Putrescin | Reagent | Sigma | P 5780 | |
| Sodium selenite | Reagent | Sigma | S 9133 | |
| Progesterone | Reagent | Sigma | P 6149 | |
| Gentamicine | Reagent | Invitrogen | ||
| L-Glutamine | Reagent | Invitrogen | 25030-024 | 200 mM (100X), liquid |
| Bovine serum albumine (BSA) | Reagent | Roth | 8076.3 | 30 mg/ml |
| Collagenase | Reagent | Sigma | C 0773 | 200 U/ml |
| Trypsin | Reagent | Sigma | T4799 | From porcine pancreas; 1 mg/ml |
| Hyaluronidase | Reagent | Sigma | H 3884 | 200 mg/ml |
| DNase I | Reagent | Roche | 1 284 932 | 10 mg/ml |
| EDTA | Reagent | Sigma | E 6511 | |
| Silicone solution | Reagent | Serva | 35130 | |
| Paraformaldehyde (PFA) | Reagent | Sigma | P6148 | 8% PFA in 0.1M phosphate buffer (pH 7.4). |
| 4′,6-diamidino-2-phenylindole dihydrochloride | Reagent | Sigma | D 0542 | DAPI |
| Fluorescent Mounting Medium | Reagent | Dako | S3023 | |
| BrDU | Reagent | Sigma | B 9285 | |
| 96-well plates | Tool | FALCON | 3072 | |
| 24-well plates | Tool | FALCON | 3047 | |
| Pasteur pipettes | Tool | Brand | 747720 | |
| Forceps DUMONT #5 | Tool | Fine Science Tools | 11252-30 | bevelled very fine shanks (0.05 mm x 0.02 mm tip) |
| Forceps DUMONT #7 | Tool | Fine Science Tools | 11271-30 | curved shanks (0.07 mm x 0.10 mm tip) |
| Spring scissors,straight, 8cm | Tool | Fine Science Tools | 15000-00 | fine, small straight blades |
| Standard scissors, straight, sharp/blunt | Tool | Fine Science Tools | 14007-14 | Use for decapitation or cervical dislocation |
| Eppendorf tubes | Tool | Eppendorf | 2ml; round bottom for better precipitation of pellet during centrifugation /cytospin | |
| Cooling centrifuge | Tool | Eppendorf | ||
| Rotation shaker | Tool | CAT | ||
| Cytospin | Tool | Thermo Scientific |
Riferimenti
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Tags
In Vivo-like Organotypic Murine Retinal Wholemount CultureTargeted Gene AblationsAnimal ModelsRetinal Gene FunctionTransgenic Mouse ModelsConditional Knockout Mouse ModelsPrimary Cell CultureRecombinant FactorsGene OverexpressionSiRNA-mediated Gene KnockdownControlled EnvironmentDissociated Cell CultureEndogenous SignalsExogenously Triggered EffectsPharmacological ManipulationOrganotypic Retinal Wholemount CulturesPhysiological In Vivo SituationNeuronal InteractionsConnections PreservedDissection PeculiaritiesEmbryonic EyesPostnatal EyesAdult Murine EyesNeuronal ApoptosisRetinal Cell Proliferation
Citazione di questo articolo
Gustmann, S., Dünker, N. In vivo-like Organotypic Murine Retinal Wholemount Culture. J. Vis. Exp. (35), e1634, doi:10.3791/1634 (2010).