脳神経細胞の初代培養の確立が続く動物の急速なジェノタイピング

Summary

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

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。

Introduction

Rodent models of genetic diseases have proven useful in establishing the physiological functions of normal proteins and nucleic acids, as well as the pathophysiological consequences of defects in these. Examples include mice deficient for proteins involved in key cellular functions, as well as mouse models of disorders such as Alzheimer's disease. However, certain genetic manipulations can lead to neonatal lethality shortly or a few days after birth. In these cases, primary cell cultures are an important tool because live cells can be obtained from the embryonic or neonatal pups before death, they can be maintained for at least a few weeks in vitro, and during this time early neuronal development can be followed by biochemical, functional and morphological experiments. For the primary cultures, it can be beneficial to plate the neurons at low density; this makes it possible to visualize the individual somata, dendrites, axonal shafts and nerve terminals at high spatial resolution. However, the survival and differentiation of neurons at low density typically requires that they are plated on a glial feeder layer, co-cultured with glial cells in the absence of physical contact with them, or cultured in medium conditioned by glia ¹.

The establishment of low-density neuronal cultures on glial feeder layers can be dependent on fast and reliable genotyping beforehand – within a few hr in contrast to a few days. Speed is especially important when the neuronal genotype needs to be matched to that of a glial feeder layer prepared beforehand. As a more practical example, it may be necessary to decide which pups of which genotype to use in generating cultures, to optimize the efficiency of an experiment.

Here we demonstrate the working protocol that has been used for fast, simplified and reliable mouse genotyping in previous publications ^2-6. Mouse tails and a commercially available kit are used. This protocol includes single-step extraction of nucleic acids from the tissue, and requires neither a nucleic-acid purification step nor use of a termination buffer ('stop solution'). The reliability of this genotyping method is illustrated by presenting the results of a series of tests when differences are introduced with respect to the starting amount of the specimens, the age of the animals and the length of the PCR amplicons. This kit offers the advantages of automated extraction and reliability.

For the sake of being comprehensive, the use of tattooing for long-term identification of the genotyped mice is also demonstrated. Tattooing is achieved by applying tattooing ink to the dermis of the skin (under the epidermis) ⁷. A procedure is described for tattooing the paw pads of newborn or 1 day old mice, although tattoos can be applied to other parts of the body, such as tails and toes, and to animals of all ages. In addition, procedures will be demonstrated for plating and culturing mouse neurons at a low density, based on optimized preparation of different types of glial feeder layers ^2,8.

We use a genetic mouse model of the inherited neurological disorder DYT1 dystonia – an autosomal-dominant movement disorder caused by a mutation in the gene TOR1A (c.904_906delGAG/c.907_909delGAG; p.Glu302del/p.Glu303del) ⁹. The encoded protein, torsinA, belongs to the “ATPases associated with diverse cellular activities” (AAA+) family of proteins, whose members generally perform chaperone-like functions, assisting in: protein unfolding, protein-complex disassembly, membrane trafficking, and vesicle fusion ^10-13. The mutation results in an in-frame deletion of a codon for glutamic acid, and can lead to manifestation of 'early-onset generalized isolated dystonia' ^14,15. However, the pathophysiological mechanisms responsible for this disorder remain poorly understood. In a knock-in mouse model, the mutant allele is Tor1a^tm2Wtd, mentioned hereafter as Tor1a^ΔE. Heterozygous ΔE-torsinA knock-in mice are viable and genetically mimic human patients with DYT1 dystonia, whereas homozygous knock-in mice die after birth ^16,17, with the latency to postnatal death affected by genetic background ¹⁸. The early death of homozygous knock-in mice necessitates that both the genotyping of animals and the establishment of neuronal cultures are completed rapidly. As another example of genotyping, Tfap2a (transcription factor AP-2α, activating enhancer binding protein 2α) will be used. The protein encoded by this gene is important in regulating multiple cellular processes, such as proliferation, differentiation, survival and apoptosis ¹⁹.

Protocol

NOTE: All animal procedures performed in this study were approved by the Institutional Animal Care and Use Committee of the University of Iowa. 1. Long-term Identification of Mice Using Tattooing the Paw Pads Immobilize a paw with the paw pad (plantar surface) facing the experimenter. Hold the paw with the thumb and the index finger. Be careful not to pinch the paw. NOTE: Stable immobilization is important to ensure that the tattoo pigment is placed into the dermis of the pa…

Representative Results

As an example of the application of this protocol, representative results are shown for labeling mice by tattooing, reliable genotyping under various experimental conditions, and establishing primary neuronal cultures on glial feeder layers. Tattooing Newborn pups were labeled on the paw pads using a tattooing system ('Newborn' in Figure 1). The labels remained clearly visible at 3 weeks ('3-week-old') and 32 weeks o…

Discussion

The protocol presented here includes procedures for tattooing to label/identify mice, for genotyping mice from tail tips, and for culturing mouse brain neurons at low density. In one round of experiments using 6-8 pups, these procedures typically require ~0.5 hr, ~4 hr and ~2 hr, respectively, at a total of 6-7 hr. This makes it practical for a single experimenter to complete all the procedures necessary from the time of the pups' birth to the plating of neuronal cultures – in less than a single working day (wi…

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