Abstract
哺乳類の神経細胞の形態および機能の高分解能分析は、多くの場合、ニューロンの初代培養物の分析を行った個々の動物の遺伝子型決定を必要とする。遺伝子型を決定する新生マウス、迅速な遺伝子型判定を標識し、そしてこれらのマウスからの脳のニューロンの低密度培養を確立する:我々はのための一連の手順について説明します。個々のマウスは、大人になって持続長期的な識別を可能に入れ墨の標識で標識されている。説明されたプロトコルによる遺伝子型決定は、高速かつ効率的で、かつ信頼性よく、核酸の自動抽出が可能になる。これは、従来の遺伝子型判定のための十分な時間が新生児致死苦しむマウスでは、例えば 、利用できない状況下で有用です。初代ニューロン培養物を、高い空間分解能で画像化実験を可能にし、低密度で生成される。この培養方法は、従来の神経めっきグリアフィーダー層の調製を必要とする。 Protocolは、運動障害DYT1ジストニア(ΔE-torsinAノックインマウス)のマウスモデルにその全体が印加され、神経細胞培養物を、これらのマウスの海馬、大脳皮質および線条体から調製される。このプロトコルは、他の遺伝子の突然変異を有するマウスに、ならびに他の種の動物に適用することができる。さらに、プロトコルの個々の成分は、単離されたサブプロジェクトのために使用することができる。したがって、このプロトコルは、神経科学ではなく、生物学的および医学科学の他の分野だけでなく、幅広い応用を持つことになります。
Materials
Name | Company | Catalog Number | Comments |
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 1,500 units/20 μl = 75,000 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 | |
EQUIPMENT - tattooing | |||
AIMS | Animal Identification and Marking Systems, Inc. | NEO–9 | This Neonate Rodent Tattooing System is an electric system that works by rapidly moving 1- or 3-point tattoo needles vertically into the skin. Activate the tattoo machine once for approximately 0.5 sec, while the tattoo needle tips are kept perpendicular to the skin surface. We prefer three-needle tattooing to maximize the tattooed area, but one-needle tattooing is effective on narrower areas, e.g. the toes, or if fine mechanical control is necessary, e.g. when numbers are tattooed. Two rounds of tattooing at the slowest speed (setting "1" out of 3 steps) are typically sufficient to produce a visible and long-lasting tattoo of the paw pads. |
EQUIPMENT - genotyping | |||
Electrophoresis system, horizontal, Wide Mini–Sub Cell GT | BIO–RAD | 170–4405 | Typical electrophoresis parameters are electrical field strength at 6 V/cm and 25 min duration for a 10 cm gel. |
FluorChem 8800 | ProteinSimple | FluorChem 8800 | |
PCR, MJ Mini Thermal Cycler | BIO-RAD | PTC-1148EDU | Our PCR reactions for the Tor1a gene in ΔE-torsinA knock-in mice are as follows: 1 cycle of denaturation at 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 30 sec, annealing at 58 °C for 30 sec, extension at 72 °C for 2 min. This is followed by final extension at 72 °C for 10 min, and holding at 4 °C. |
Power supply, PowerPac Basic | BIO-RAD | 164-5050 | |
EQUIPMENT - cell culture | |||
Automated cell counter, Countess | GIBCO-Invitrogen | C10310 | This automated cell counter separately measures the densities of live and dead cells (non stained and stained by trypan blue, respectively). It is important to know the optimal range of density measurements: the counter that we use has the highest accuracy in the range from 1 x 105 to 4 x 106 cells/ml. If the measured cell density values fall outside the recommended range, adjust the resuspension volume appropriately. |
Biological Safety Cabinet, Class II, Type A2 | NUAIRE | NU-425-400 | This hood is used for all cell culture procedures, except for brain dissection. |
CO2 Incubator, AutoFlow, Humidity Control Water Jacket | NUAIRE | NU-4850 | |
Horizontal Clean Bench | NUAIRE | NU-201-330 | This clean bench is used for brain dissection (steps 3.1.1 and 3.1.2 of "Brain Dissection and Cellular Dissociation)". |
Orbit LS Low Speed Shaker | Labnet | S2030-LS-B | |
SORVALL RC-6 Plus Superspeed Centrifuge | Fisher | 46910 (centrifuge)/46922 (rotor) |
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