基因传递到产后大鼠脑非心室质粒注射和电穿孔
Summary
这个协议描述了一种非病毒的基因结构的交付方法的生活啮齿动物脑的某些区域。该方法由质粒制备，微管制造，新生鼠的小狗手术，显微注射的构造，并<em>在体内</em>电。
Abstract
转基因动物的创作是一种标准的方法，在研究一个基因在体内的兴趣的功能。然而，许多基因敲除或转基因动物是没有生命力的，在这些修改后的基因表达或在整个有机体中删除的情况下。此外，各种补偿机制往往使人们难以解释的结果。补偿效果，无论是定时的基因表达或转染细胞的数量限制，可缓解。
产后非心室显微注射和体内电的方法，可以有针对性地提供基因，siRNA或染料分子直接到一个小区域的新生啮齿动物脑的兴趣。在传统的心室注射技术相比，这种方法允许非洄游类型的细胞转染。这里介绍的方法转染的动物，可用于双光子，例如，在活体成像对急性脑片电生理实验。
Protocol
1。简介转基因动物的创作是一种强大的方法，在动物活体1，解开疾病机制2,3以及操纵细胞的属性4的基因的功能进行调查。但是，该过程是相当费力，非常耗时且昂贵，因此有必要使用替代的基因传递方法，如病毒注射液5， 电 6和新生儿在子宫内电 7,8脑室注射。产后非室注射和电的方法，都有一套独特的优势：使用非?…
Discussion
到生活的啮齿类动物的大脑中的基因传递方法是建立在子宫内电7,8,11,12和，最近， 产后电6。然而，这些方法的基础上脑室注射质粒DNA，这可能是多个应用程序的限制。例如，这些方法不容许针对某些脑区如海马，也不等非迁徙作为皮层星形胶质细胞细胞类型的转染的细胞。首先被用于基因运送到13小脑的神经元电耦合的非室注射。我们的协议说明非心室法对大鼠脑…
Acknowledgements
我们感谢配乐录制的视频，伊万Molotkov为CAG – EGFP质粒制备的三维动画和彼得Blaesse博士的帮助，叶卡捷琳娜Karelina。
这项工作是由来自芬兰的国际流动，芬兰文化基金会和芬兰科学院中心的赠款支持。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 2A-sa dumb Tweezers, 115mm | equipment | XYtronic | XY-2A-SA | Treat with 70% ethanol for disinfection before use in surgical manipulations |
| Biological Temperature Controller with stainless steel heating pad | equipment | Supertech | TMP-5b | |
| Borosilicate tube with filament | material | Sutter Instruments | BF120-69-10 | Glass needle |
| Disposable drills | material | Meisinger | HP 310 104 001 001 008 | |
| Dulbeco’s PBS 10X | reagent | Sigma | D1408 | |
| Dumont #5 forceps, 110 mm | equipment | FST | 91150-20 | Treat with 70% ethanol for disinfection before use in surgical manipulations |
| Ealing microelectrode puller | equipment | Ealing | 50-2013 | Vertical electrode glass puller |
| Ethilon monofil polyamide 6-0 FS-3 16 mm 3/8c | material | Johnson & Johnson Medical | EH7177H | Surgical threads |
| Exmire micro syringe 10.0 ml | equipment | Exmire | MS*GLLX00 | Gas-tight syringe |
| Fast Green | reagent | Sigma | F7252 | |
| Forceps electrodes | equipment | BEX | LF650P3 | Treat with 70% ethanol for disinfection prior to use |
| Foredom drill control | equipment | Foredom | FM3545 | Surgical drill power supply and control. Currently available analogue is micromotor kit K.1070 (Foredom) |
| Foredom micro motor handpiece | equipment | Foredom | MH-145 | Currently available analogue is micromotor kit K.1070 (Foredom) |
| Gas anesthesia platform for mice | equipment | Stoelting | 50264 | Assembled on stereotaxic instrument |
| Isoflurane | reagent | Baxter | FDG9623 | |
| Micro dressing forceps, 105 mm | equipment | Aesculap | BD302R | Treat with 70% ethanol for disinfection before use in surgical manipulations |
| Microfil | material | WPI | MF34G-5 | Micro syringe filling capillaries |
| Mineral oil | reagent | Sigma | M8410 | |
| NanoFil Syringe 10 microliter | equipment | WPI | NANOFIL | Hamilton syringe |
| plasmid CAG-EGFP | reagent | Extracted and purified with EndoFree Plasmid Maxi Kit (Qiagen) and dissolved in nuclease free water to concentration 1.5 mg/ml | ||
| Pulse generator CUY21Vivo-SQ | equipment | BEX | CUY21Vivo-SQ | |
| Schiller electrode gel | reagent | Schiller AG | 2.158000 | Conductive gel |
| Small animal stereotaxic instrument | equipment | David Kopf Instruments | 900 | |
| Stoelting mouse and neonatal rat adaptor | equipment | Stoelting | 51625 | Assembled on stereotaxic instrument. Treat earbars with 70% ethanol for disinfection before use in surgical manipulations |
| Student iris scissors, straight 11.5 cm | equipment | FST | 91460-11 | Treat with 70% ethanol for disinfection before use in surgical manipulations |
| Sugi absorbent swabs 17 x 8 mm | material | Kettenbach | 31602 | Surgical tampons |
| UMP3 microsyringe pump and Micro 4 microsyringe pump controller | equipment | WPI | UMP3-1 | Microinjector and controller |
| Univentor 400 Anesthesia Unit | equipment | Univentor | 8323001 |
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Tags
Gene DeliveryPostnatal Rat BrainNon-ventricularPlasmid InjectionElectroporationTransgenic AnimalsGene Of InterestKnockout AnimalsCompensatory MechanismsGene Expression TimingTransfected CellsMicroinjectionIn Vivo ElectroporationTargeted DeliverySiRNADye MoleculesNewborn Rodent BrainNon-migratory Cell TypesTwo-photon In Vivo ImagingElectrophysiological Experiments
Citazione di questo articolo
Molotkov, D. A., Yukin, A. Y., Afzalov, R. A., Khiroug, L. S. Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation. J. Vis. Exp. (43), e2244, doi:10.3791/2244 (2010).