动物的基因分型快速其次是建立大脑神经元原代培养
Summary
我们描述了标签和基因分型新生小鼠，并从中产生的主要神经文化的过程。基因分型是快速，有效和可靠的，并且允许进行自动核酸提取。这是为neonatally致死小鼠和其文化，需要事先完成基因分型的特别有用的。
Abstract
哺乳动物神经元的形态和功能的高分辨率分析往往需要个体动物随后的神经元的原代培养的分析的基因分型。我们描述了一套程序：标记新生小鼠进行基因分型，快速基因分型，并建立从这些小鼠脑的神经元的低密度培养物。个别小鼠通过纹身，其允许长期持久的识别到成年标记。基因分型通过所述协议是快速和有效的，并且允许进行核酸具有良好的可靠性的自动提取。这是根据情况下有足够的时间对常规的基因分型是不可用， 例如 ，是有用的，在于从新生致死遭受小鼠。在低浓度，这使得成像实验以高的空间分辨率产生原代神经元培养物。这种培养方法需要胶质滋养层的准备之前，神经元电镀。在protocol是其全部施加到运动障碍DYT1肌张力障碍（ΔE-torsinA敲除小鼠）的小鼠模型中，与神经元培养物从这些小鼠的海马，大脑皮质和纹状体制备。该协议可以被应用到小鼠与其他的基因突变，以及对其他物种的动物。此外，可用于分离子项目的协议的各个组件。因此，该协议将有广泛的应用，不仅在神经科学，而且在生物科学和医学等领域。
Introduction
遗传疾病的啮齿动物模型已被证明在建立正常蛋白质和核酸，以及在这些缺陷的病理生理后果的生理功能非常有用。举出缺陷型为参与关键的细胞功能的蛋白质的小鼠，以及病症如阿耳茨海默氏病的小鼠模型。然而，某些遗传操作可导致新生儿杀伤力不久或在出生后几天。在这些情况下，原代细胞培养物，因为活细胞可以从死亡前的胚胎或新生儿幼仔得到的重要工具，它们可以保持在至少几个星期的体外 ，并在这段时间内的早期神经元发育，可接着生化，功能和形态的实验。对于主要培养物，也可以是有益的直接制版的神经元密度低;这使得可以以可视化的个别胞体，树突，轴突轴和神经TERMINALS在高的空间分辨率。然而，神经元在低浓度的存活和分化通常要求它们被镀上一个胶质饲养层，共培养的神经胶质细胞在不存在与它们由胶质1空调物理接触，或在培养基中培养。
低密度的神经元培养物的对神经胶质饲养层的机构可以是依赖于快速和可靠的基因分型预先 – 在几个小时在对比几天。速度是特别重要的神经基因型需要被匹配到预先制备的胶质饲养层时。作为更实际的例子，它可能有必要决定哪些其中基因型的幼仔在产生培养物的使用，以优化实验的效率。
在这里，我们证明了工作的协议，已被用于快速，简单和可靠的小鼠基因分型在以前的出版物2-6。鼠标的尾巴，市售的试剂盒的使用。这个协议包括从组织单步提取核酸，并且既不需要一个核酸纯化步骤，也不使用终止缓冲液（“停止溶液”）。这种基因分型方法的可靠性是通过提出的一系列试验的结果时，差异被相对于该样品的起始量，动物的年龄和PCR扩增子的长度引入的说明。该套件提供了自动提取和可靠性的优势。
为求是全面，使用纹身长期鉴定基因型小鼠的也证实。纹身是通过将纹身油墨对皮肤的真皮（表皮下）实现7。的过程被描述为纹身新生儿或1日龄小鼠的爪垫，虽然纹身可以应用到身体的其他部位，如尾和脚趾，并以动画所有年龄段的ALS。另外，程序将被证明为在低密度电镀和培养小鼠的神经元，是根据不同类型的神经胶质饲养层2,8的优化制剂。
我们使用继承神经系统疾病DYT1肌张力障碍的遗传小鼠模型-造成的基因TOR1A突变为常染色体显性运动障碍（c.904_906delGAG / c.907_909delGAG; p.Glu302del / p.Glu303del）9。所编码的蛋白质，torsinA，属于（AAA +）蛋白质家族，其成员通常执行伴侣样的功能，帮助在“与不同的细胞活性相关的ATP酶”：蛋白质折叠，蛋白质复合物的拆卸，膜运输，和囊泡融合10-13。该突变导致一个在框内缺失谷氨酸的密码子的，并可能导致“早发性全身性肌张力障碍分离的”14,15的表现。但是，路径负责这种疾病ophysiological机制仍然知之甚少。在一个敲入小鼠模型中，该突变体等位基因是TOR1A tm2Wtd，以下简称TOR1AΔE提及。杂ΔE-torsinA基因敲除小鼠是可行和基因模仿人类患者的肌张力障碍DYT1，而纯合子基因敲除小鼠出生后16,17死，与延迟到出生后死亡受遗传背景的18。纯合敲除小鼠的早期死亡的必要条件是动物两者的基因分型，并建立的神经元培养物的迅速完成。作为分型的另一个例子，Tfap2a（转录因子AP-2α，活化增强子结合蛋白2α）将被使用。由该基因编码的蛋白质是在调节多种细胞过程，诸如增殖，分化，存活和凋亡19重要。
Protocol
注：在本研究中进行的所有动物的程序批准了美国爱荷华大学的机构动物护理和使用委员会。 利用小鼠的纹身爪垫1.长期鉴定固定用爪子垫（足底面）一爪子对着实验者。持用拇指和食指的爪子。要小心，不要捏爪子。 注：稳定的固定化是很重要的，以确保该纹身颜料放入爪垫的真皮，并因此是永久性7。 拭爪垫，用70％的乙醇在纱布海绵或拭子。 …
Representative Results
作为该协议的应用的一个例子，有代表性的结果示于通过纹身，可靠的基因分型的各种实验条件下标记的小鼠，并且对神经胶质饲养层建立初级神经元培养物。 纹身 新生幼仔被标记上（ 图1中“新生儿”），使用纹身系统的爪垫。标签保持3周清晰可见（'3周龄'）和32周龄（'32周龄'）。个别小鼠可通过在四只爪子（编…
Discussion
这里介绍的协议包括程序纹身标记/识别小鼠的基因分型，从尾部的提示小鼠，并为培养小鼠大脑的神经元密度低。在一个循环中使用6-8幼仔的实验中，这些过程通常需要约0.5小时，〜4小时和〜2小时，分别在共6-7小时的。这使得实际用于单个实验者完成所有从幼仔的出生到神经元培养物的电镀时所必需的程序 – 在少于单个工作日（除胶质饲养层之前制备的）。
纹身 </p…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors thank researchers at the University of Iowa, Drs. Luis Tecedor, Ines Martins and Beverly Davidson for instructions and helpful comments regarding striatal cultures, and Drs. Kara Gordon, Nicole Bode and Pedro Gonzalez-Alegre for genotyping assistance and discussions. We also thank Dr. Eric Weyand (Animal Identification and Marking Systems) for helpful comments regarding tattooing, and Dr. Shutaro Katsurabayashi (Fukuoka University) for helpful comments regarding the mouse culture. This work was supported by grants from the American Heart Association, the Department of Defense (Peer Reviewed Medical Research Program award W81XWH-14-1-0301), the Dystonia Medical Research Foundation, the Edward Mallinckrodt, Jr. Foundation, the National Science Foundation, and the Whitehall Foundation (N.C.H.).
Materials
| REAGENTS – tattooing | |||
| Machine Cleanser | Animal Identification and Marking Systems, Inc. | NMCR3 | This is used to clean the needles and the holder after tattooing. |
| Machine Drying Agent | Animal Identification and Marking Systems, Inc. | NDAR4 | This is used to dry the needles and holder after cleaning. |
| Neonate Tattoo Black Pigment | Animal Identification and Marking Systems, Inc. | NBP01 | |
| Skin Prep Applicator | Animal Identification and Marking Systems, Inc. | NSPA1 | Q-tip. |
| Skin Prep solution | Animal Identification and Marking Systems, Inc. | NSP01 | This reagent delivers a thin layer of oil that enhances the efficiency of tattooing and prevents tattoo fading, by (information from vendor): 1) preventing non-tattooed skin from being stained temporarily, thereby allowing the quality of a paw pad tattoo to be easily evaluated before the pup is returned to its home cage – the stained skin surface can be confused with the tattooed skin, 2) reducing skin damage during tattooing – softening the skin and lubricating the needle will help the needle penetrate the skin without causing skin damage, and 3) preventing molecular oxygen from entering the skin, thereby reducing inflammatory responses to reactive oxygen species that can be generated. |
| REAGENTS – genotyping | |||
| EZ Fast Tissue/Tail PCR Genotyping Kit (Strip Tube Format) | EZ BioResearch LLC | G2001-100 | |
| 2X PCR Ready Mix II | EZ BioResearch LLC | G2001-100 | A red, loading dye for electrophoresis is included in the 2X PCR Ready Mix solution. |
| Tissue Lysis Solution A | EZ BioResearch LLC | G2001-100 | Prepare DNA Extraction Solution by mixing 20 µl of Tissue Lysis Solution A and 180 µl of Tissue Lysis Solution B per specimen. |
| Tissue Lysis Solution B | EZ BioResearch LLC | G2001-100 | Prepare DNA Extraction Solution by mixing 20 µl of Tissue Lysis Solution A and 180 µl of Tissue Lysis Solution B per specimen. |
| Acetic acid, glacial | VWR | BDH 3092 | |
| Agarose optimized grade, molecular biology grade | rpi | A20090-500 | We use 2% agarose gels in TAE buffer containing the SYBR Safe DNA gel stain (diluted 10,000-fold) or ethidium bromide (0.5 µg/ml gel volume). |
| Ethidium bromide | Sigma-Aldrich | E7637-1G | |
| Ethylenediamine tetraacetic acid, disodium salt dihydrate (EDTA) | Fisher | BP120-500 | |
| Filtered Pipet Tips, Aerosol-Free, 0.1-10 µl | Dot Scientific Inc | UG104-96RS | Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination. |
| Filtered Pipet Tips, Premium Fit Filter Tips, 0.5-20 µl | Dot Scientific Inc | UG2020-RS | Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination. |
| Filtered Pipet Tips, Premium Fit Filter Tips, 1-200 µl | Dot Scientific Inc | UG2812-RS | Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination. |
| Molecular weight marker, EZ DNA Even Ladders 100 bp | EZ BioResearch LLC | L1001 | We use either of these three molecular weight markers. |
| Molecular weight marker, EZ DNA Even Ladders 1000 bp | EZ BioResearch LLC | L1010 | |
| Molecular weight marker, TrackIt, 100 bp DNA Ladder | GIBCO-Invitrogen | 10488-058 | |
| PCR tubes, 8-tube strips with individually attached dome top caps, natural, 0.2 ml | USA Scientific | 1402-2900 | Use tubes that are sterile and free of DNA, RNase and DNase. An 8-tube strip is easy to handle and to group the specimens than individual tubes. |
| PCR tubes, Ultraflux Individual | rpi | 145660 | Use tubes that are sterile and free of DNA, RNase and DNase. |
| Seal-Rite 0.5 ml microcentrifuge tube, natural | USA Scientific | 1605-0000 | Use tubes that are sterile and free of DNA, RNase and DNase. |
| SYBR Safe DNA gel stain * 10,000x concentration in DMSO | GIBCO-Invitrogen | S33102 | |
| Tris base | rpi | T60040-1000 | |
| Primers for amplifying Tor1a gene in ΔE-torsinA knock-in mice | 5'-AGT CTG TGG CTG GCT CTC CC-3' (forward) and 5'-CCT CAG GCT GCT CAC AAC CAC-3' (reverse) (reference 18). These primers were used at a final concentration of 1.0 ng/µl (~0.16 µM) (reference 2). | ||
| Primers for amplifying Tfap2a gene in wild-type mice | 5'-GAA AGG TGT AGG CAG AAG TTT GTC AGG GC-3' (forward), 5'-CGT GTG GCT GTT GGG GTT GTT GCT GAG GTA-3' (reverse) for the 498-bp amplicon, 5'-CAC CCT ATC AGG GGA GGA CAA CTT TCG-3' (forward), 5'-AGA CAC TCG GGC TTT GGA GAT CAT TC-3' (reverse) for the 983-bp amplicon, and 5'-CAC CCT ATC AGG GGA GGA CAA CTT TCG-3' (forward), 5'-ACA GTG TAG TAA GGC AAA GCA AGG AG-3' (reverse) for the 1990-bp amplicon. These primers are used at 0.5 µM. | ||
| REAGENTS – cell culture | |||
| 5-Fluoro-2′-deoxyuridine | Sigma-Aldrich | F0503-100MG | See comments section of uridine for more information. |
| B-27 supplement | GIBCO-Invitrogen | 17504-044 | |
| Cell Culture Dishes 35 x 10 mm Dishes, Tissue Culture-treated | BD falcon | 353001 | |
| Cell Culture Flasks, T25, Tissue Culture-treated, Canted-neck, plug-seal cap, 25 cm2 Growth Area, 70 ml | BD falcon | 353082 | |
| Cell Culture Flasks, T75, Tissue Culture-treated, Canted-neck, vented cap, 75 cm2 Growth Area, 250 ml | BD falcon | 353136 | |
| Conical Tube, polypropylene, 15 ml | BD falcon | 352095 | |
| Countess (cell number counter) chamber slides | GIBCO-Invitrogen | C10312 | |
| Cytosine β-D-Arabinofuranoside hydrochloride (Ara-C hydrochloride) | Sigma-Aldrich | C6645-100mg | |
| D-(+)-Glucose (Dextrose) anhydrous, SigmaUltra, 99.5% (GC) | Sigma-Aldrich | G7528-250G | |
| Dish, Petri glass 100 x 15 mm | Pyrex | 3160-101 | |
| Distilled water | GIBCO-Invitrogen | 15230-147 | |
| DNase Type II | Sigma-Aldrich | D4527-200KU | Stock solution is prepared at 1500 units/20 μl = 75000 units/ml in distilled water. |
| Dulbecco's Modified Eagle Medium (DMEM), high glucose, GlutaMAX, pyruvate | GIBCO-Invitrogen | 10569-010, 500 ml | |
| Fast PES Filter Unit, 250 ml, 50 mm diameter membrane, 0.2 µm Pore Size | Nalgene | 568-0020 | |
| Fast PES Filter Unit, 500 ml, 90 mm diameter membrane, 0.2 µm Pore Size | Nalgene | 569-0020 | |
| Fetal bovine serum (FBS) | GIBCO-Invitrogen | 26140-079 | |
| Glass coverslip, 12 mm Round, thickness 0.09–0.12 mm, No. 0 | Carolina | 633017 | |
| GlutaMAX-I | GIBCO-Invitrogen | 35050-061 | |
| Hanks' Balanced Salts | Sigma-Aldrich | H2387-10X | |
| HEPES, ≥99.5% (titration) | Sigma-Aldrich | H3375-250G | |
| Hydrochloric acid, 37%, A.C.S reagent | Sigma-Aldrich | 258148-100 ML | |
| Insulin | Sigma-Aldrich | I5500-250 mg | |
| Magnesium sulfate heptahydrate, MgSO4•(7H2O), BioUltra, ≥99.5% (Fluka) | Sigma-Aldrich | 63138-250G | |
| Matrigel Basement Membrane Matrix solution, Phenol Red-Free | BD Biosciences | 356237 | This is the coating material for coverslips and flasks. 1) To prepare it, thaw the Matrigel Basement Membrane Matrix solution on ice, which usually takes ~1 day. Using a pre-cooled pipette, aliquot the thawed solution into pre-cooled T25 flasks on ice, and store the flasks at -20°C. To prepare the working Matrigel solution, thaw the aliquotted Matrigel in a flask on ice, dilute 50-fold by adding pre-cooled MEM solution and keep the diluted solution at 4°C. It is important to pre-cool all cultureware and media that come into contact with Matrigel, except during and after the coating of coverslips, to prevent it from prematurely forming a gel. 2) To coat the glass coverslips or culture flasks with Matrigel, apply the Matrigel solution to the surface. Before plating cells, it is important to completely dry up the surface. For this purpose, it might be helpful to aspirate Matrigel during the cellular centrifugation immediately before plating the cells and to allow enough time for drying. |
| Minimum Essential Medium (MEM) | GIBCO-Invitrogen | 51200-038 | |
| MITO+ Serum Extender, 5 ml | BD Biosciences | 355006 | |
| Multiwell Plates, Tissue Culture-treated 24-well plate | BD falcon | 353047 | |
| Multiwell Plates, Tissue Culture-treated 6-well plate | BD falcon | 353046 | |
| Neurobasal-A Medium (1X), liquid | GIBCO-Invitrogen | 10888-022 | |
| Nitric Acid | VWR | bdh 3044 | |
| NS (Neuronal Supplement) 21 | prepared in the lab | Source: reference 69 | |
| Pasteur pipets, 5 ¾” | Fisher | 13-678-6A | Use this cotton-plugged 5 ¾” Pasteur pipette for cellular trituration. Fire-polish the tip beforehand to smooth the cut surface and to reduce the internal diameter to 50-80% of the original. Too small a tip will disrupt the cells and reduce cell viability, but too large a tip will decrease the efficiency of trituration. |
| Pasteur pipets, 9” | Fisher | 13-678-6B | |
| Potassium chloride (KCl), SigmaUltra, ≥99.0% | Sigma-Aldrich | P9333-500G | |
| Serological pipet, 2 ml | BD falcon | 357507 | |
| Serological pipet, 5 ml | BD falcon | 357543 | |
| Serological pipet, 10 ml | BD falcon | 357551 | |
| Serological pipet, 25 ml | BD falcon | 357525 | |
| Serological pipet, 50 ml | BD falcon | 357550 | |
| Sodium bicarbonate (NaHCO3, Sodium hydrogen carbonate), SigmaUltra, ≥99.5% | Sigma-Aldrich | S6297-250G | |
| Sodium chloride (NaCl), SigmaUltra, ≥99.5% | Sigma-Aldrich | S7653-250G | |
| Sodium hydroxide (NaOH), pellets, 99.998% trace metals basis | Sigma-Aldrich | 480878-250G | |
| Sodium phosphate dibasic heptahydrate (Na2HPO4•(7H2O)), ≥99.99%, Aldrich | Sigma-Aldrich | 431478-250G | |
| Sucrose, SigmaUltra, ≥99.5% (GC) | Sigma-Aldrich | S7903-250G | |
| Syringe filter, sterile, 0.2 µm | Corning | 431219 | |
| Syringe, 3 ml | BD falcon | 309585 | |
| Transferrin, Holo, bovine plasma | Calbiochem | 616420 | |
| Trypan Blue stain, 0.4% | GIBCO-Invitrogen | T10282 | This is used for counting live/dead cells. Renew an old trypan blue solution if it is re-used many times (e.g. several times a week for several weeks), because it will form precipitates and result in erroneous readouts of cellular density. |
| Trypsin, type XI | Sigma-Aldrich | T1005-5G | |
| Trypsin-EDTA solution, 0.25% | GIBCO-Invitrogen | 25200-056 | |
| Uridine | Sigma-Aldrich | U3003-5G | Stock solution is prepared at 50-mg 5-fluoro-2'-deoxyuridine and 125-mg uridine in 25 ml DMEM (8.12 and 20.48 mM, respectively). |
| REAGENTS – immunocytochemistry | |||
| Antibody, rabbit polyclonal anti-MAP2 | Merck Millipore | AB5622 | |
| Antibody, mouse monoclonal anti-GFAP cocktail | Merck Millipore | NE1015 |
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Koh, J., Iwabuchi, S., Huang, Z., Harata, N. C. Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons. J. Vis. Exp. (95), e51879, doi:10.3791/51879 (2015).