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Method Article

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

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DOI:

10.3791/51879

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January 29th, 2015

 ,  ,  , 
1Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine, 2Department of Psychiatry, University of Iowa Carver College of Medicine, 3EZ BioResearch LLC

* These authors contributed equally

In This Article

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

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

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 Tor1atm2Wtd, mentioned hereafter as Tor1aΔE. 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.

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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

  1. 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 paw pad, and thus is permanent 7.
  2. Swab the paw pad with 70% ethanol on a gauze sponge or swab.
  3. Apply skin oil to a cotton-tipped applicator, and gently press the tip against the surface of the paw pad several times. Use only a small amount of skin oil; when present in large amounts, it will prevent the tattoo pigment from reaching the skin.
  4. Dip the tips of the tattoo needles into the tattooing ink just prior to tattooing. Use clean, aseptic and sharp tattooing needles, to decrease the pain and the possibility of infection, and also to increase the tattooing efficiency.
  5. While the paw pad surface is covered with the skin oil, press the tattoo needle tips vertically and lightly against the skin in the center of the paw pad, and inject the ink multiple times. See Table of Materials/Equipment for information about electric tattooing system.
  6. Spray 70% ethanol on a gauze sponge, gently press it against the paw to remove extra tattoo pigment on the skin surface, and inspect the quality of tattoo. Check that the middle of paw pad has a dark, round spot that differs from normal skin pigmentation. If the tattoo is not dark or large enough for easy viewing, repeat the prior tattooing steps.

2. Genotyping Newborn Mice Using a Fast PCR Genotyping Kit

  1. Disinfect the distal end of a mouse tail with 70% ethanol, cut 5 mm or less of the tail tip and transfer it to a tube of an 8-tube strip of the type used for PCR. Use an un-used razor blade for each pup to avoid cross contamination between specimens. Alternatively, use a pair of scissors, but in this case, carefully and thoroughly remove the remaining tissue on the blades using 70% ethanol. Check for bleeding. If bleeding occurs, apply pressure to the cut portion of the tail with a gauze sponge until bleeding has stopped.
  2. Add 200 µl of DNA Extraction Solution to each PCR tube containing a specimen. See Table of Materials/Equipment for its composition and information about the kit.
  3. Place the tube strip into a PCR thermal cycler, and start the DNA extraction using the following program: 1 cycle at 55 °C for 10 min, 1 cycle at 95 °C for 10 min, and holding at 4 °C.
    NOTE: This is the same PCR thermal cycler that is later used for PCR.
  4. After the DNA extraction is finished, remove the tube strip from the thermal cycler and invert 5 times.
  5. Transfer 4 µl of the solution (DNA extract) of each specimen to an un-used tube of an 8-tube strip, and mix with: 10 µl of 2X PCR Ready Mix II, 2 µl of mixed forward & reverse primers (recommended at 0.5 µM; see Table of Materials/Equipment for sequences), and 4 µl of nuclease-free H2O, for each specimen. Spin briefly (e.g., 3 sec) using a table-top centrifuge. Keep the tubes on ice at all times except when handling.
  6. Perform thermal cycling. See Table of Materials/Equipment for the thermal program.
  7. Detect the amplified DNA products. Load the total reaction solution from the PCR (20 µl) directly into a well of an agarose gel. Load the molecular weight markers into a separate well. Apply an electric field.
  8. Acquire fluorescence images of the bands under ultraviolet light.

3. Primary Culture of Mouse Brain Neurons on Glial Feeder Layer

NOTE: The procedures for brain dissection and cell dissociation (3.1) are common to all the subsequent procedures. The procedures for mouse glial cultures (3.2), rat glial cultures (3.3), and mouse neuronal cultures (3.4) are described separately afterwards.

  1. Brain Dissection and Cellular Dissocaiation
    1. Sacrifice one mouse or rat pup by decapitation, rapidly remove the brain, and place it into Hanks' solution (see Table 1 for composition) + 20% fetal bovine serum (FBS) in a 35 mm dish (kept on ice).
    2. Cut the brain through the midline into two hemispheres. Remove the region of interest(e.g., cerebral cortex, striatum and hippocampus) from each hemisphere of the brain. Remove the meninges and major blood vessels from the surface. Cut the brain region into 4-10 thin slices using a surgical blade.
    3. Rinse the brain slices in a 15 ml centrifugation tube, once with Hanks' solution + 20% FBS, then 3 times with the Hanks' solution (i.e., without serum) (all at 4 °C). To rinse, add 5-10 ml solution, let the brain slices settle at the bottom of the tube, aspirate the solution from the top, and add a fresh solution. Finish the rinsing procedure by aspirating the solution.
    4. Filter 2 ml of the trypsin-containing digestion solution (see Table 1 for composition) using a 3 ml syringe and a 0.2 µm syringe filter, and add the filtrate directly into the tube containing the brain slices. Let the trypsinization proceed for 13 min at RT.
    5. Neutralize the trypsin solution first by aspirating most of it and then by adding 7-10 ml of Hanks' solution + 20% FBS (4 °C).
    6. Rinse the brain slices, twice with Hanks' solution + 20% FBS, and then three times with the Hanks' solution (i.e. without serum) (all at 4 °C). Finish the rinsing procedure by aspirating the solution.
    7. Filter 2 ml of the dissociation solution (see Table 1 for composition) using a 3 ml syringe and a 0.2 μm syringe filter, and add the filtrate directly to the tube with the brain slices.
    8. Mechanically dissociate the cells by gently triturating 10-20 times, until visible tissue pieces disappear. Use a cotton-plugged, fire-polished Pasteur pipette and avoid making bubbles during trituration.
    9. Wait 3 min for small pieces to settle down.
    10. Transfer the majority of the solution (~1.5 ml, leaving some solution at the bottom) to a 15 ml centrifugation tube that contains 3 ml Hanks' solution + 20% FBS solution (4 °C), using the cotton-plugged, fire-polished Pasteur pipette. Do not transfer all the solution because the inclusion of any sediment at the bottom typically results in deterioration of the culture.
    11. Centrifuge for 13 min at ~185 g (~1,100 rpm) at 4 °C.
    12. Aspirate the supernatant gently, add 1 ml of pre-warmed plating medium (37 °C) to the pellet, and resuspend it by gently pipetting several times using the cotton-plugged, fire-polished Pasteur pipette.
      NOTE: Three different types of plating media are used for different culture purposes: plating medium-1 for mouse glial cells, plating medium-2 for mouse neurons, and plating medium-3 for rat neurons and glial cells (see Table 1 for compositions).
    13. Take out 10 μl of the cell suspension, mix it with 10 μl of 0.4% trypan blue solution, and measure the density of live cells, using either a hemocytometer or an automated cell counter.
  2. Mouse Glial Cultures
    1. Wash the coverslips to help establish healthy cultures of mouse cells.
      1. Immerse glass coverslips (round, 12 mm diameter) in 70% nitric acid in a glass Petri dish. Protect from light using aluminum foil, and place it on an orbital shaker O/N.
      2. Rinse the glass coverslips in the Petri dish at least three times with distilled water. Immerse them in distilled water. Place the dish on an orbital shaker O/N.
      3. Dry the coverslips on a Whatman 150 mm filter paper in the biological safety cabinet.
      4. Autoclave the coverslips.
      5. Place the coverslips into 24-well culture dish.
    2. Label and genotype newborn mice according to steps 1 and 2.
    3. Obtain the brain cells from the mouse pups, according to step 3.1.
      NOTE: Use of the cerebral cortex is typical for preparing the mouse glial feeder layer. However, other brain regions, such as hippocampus and striatum, will work after proper adjustment of cell density due to different numbers and yields of cells from different regions.
    4. Add ~4 ml of pre-warmed plating medium-1 to the final cell suspension (~1 ml). For pre-warming culture media, place the solution in the culture incubator (5% CO2-95% O2, 37 °C) O/N, to allow the temperature and pH values to stabilize.
    5. Transfer the cell suspension to an uncoated T25 culture flask, using the cotton-plugged, fire-polished Pasteur pipette. Place the flask in the culture incubator.
    6. At 1 day in vitro (DIV), rinse the cultured cells in the T25 flask twice with plating medium-1 (4 °C). Place the flask back into the incubator. Perform rinsing by aspirating the medium inside the flask completely with a Pasteur pipette, adding ~5 ml of fresh medium and gently tilting the flask several times in a swirling motion.
    7. At 6-9 DIV, (i.e., one day before trypsinization and plating on coverslips, in steps 3.2.8-3.2.13), place 100 µl of the coating material with extracellular matrix proteins (see Table of Materials/Equipment for comments about the coating material) on the glass coverslips in a culture dish, and place the culture dish in the culture incubator.
    8. At 7-10 DIV, when the cells are 20-40% confluent (spatially continuous), trypsinize them.
      1. Rinse the flask once, by aspirating all the solution within the flask and adding ~13 ml of Hanks' solution (4 °C), gently tilt the flask several times in a swirling motion, and aspirate the solution completely.
      2. Add 40 µl of DNase solution (final concentration, 750 units/ml) to 4 ml Trypsin-EDTA solution, pass the solution through a 0.2 µm syringe filter, and add the filtrate directly to the cells in the flask.
      3. Let the trypsinization proceed for 13 min at 37 °C in the incubator.
      4. Neutralize the trypsin solution by adding 2 ml of 100% FBS (4 °C) to the flask.
      5. Transfer the trypsinized cells to a 15 ml centrifugation tube using a 5- to 10-ml pipette, add ~4 ml of Hanks' solution + 20% FBS (4 °C), centrifuge at ~185 g and 4 °C for 13 min, and aspirate the supernatant.
    9. Resuspend the pellet in 1 ml of pre-warmed plating medium-1.
    10. Measure the density of the cells according to step 3.1.13.
    11. Aspirate the coating material completely from the glass coverslips, and plate ~50 μl of the resuspended glial cells on coverslips.
      NOTE: These cells will establish the glial feeder layer.
    12. Place the culture dish with coverslips in the incubator.
    13. 20-60 min later, add 1 ml of pre-warmed plating medium-1 to each well, and place the dish back in the incubator. Note: While the plating medium is added, it will be helpful to use another pipette to press down the periphery of the coverslip (where there are no plated cells), so that the coverslip will not float in the medium.
    14. At 1 DIV of the glial feeder layer (i.e., on the glass coverslip), replace the medium with 1 ml of pre-warmed plating medium-1, by aspirating all of the solution in a well and then filling it with fresh medium.
    15. At 2-3 DIV of the glial feeder layer when the cells are 80-100% confluent, add mitotic inhibitor (10 µl of a mixture of 5-fluoro-2'-deoxyuridine + uridine; final concentrations of 81.2 and 204.8 µM, respectively) to each well, to inhibit DNA replication and therefore to suppress glial-cell proliferation.
    16. At 7-9 DIV, when the cells are 90-100% confluent (1-2 hr before plating neurons on the glial feeder layer), replace the culture medium with pre-warmed plating medium-2 (37 °C).
      NOTE: Neurons will be plated on the same day, using step 3.4.
  3. Rat Glial Cultures
    1. Coat a T25 culture flask with the same coating material.
      NOTE: This is necessary for later removal of non-adherent cells in step 3.3.5.
      1. Add 2.0 ml of the coating material to the flask, and place the flask in the culture incubator.
      2. After 2-3 hr, aspirate the coating material completely, such that the floor of the flask becomes dry.
    2. Obtain the cells from the rat pups, according to step 3.1.
      NOTE: The CA3-CA1 region of the hippocampus is used for preparing the rat glial feeder layer. However, other brain regions, such as the cerebral cortex and striatum, will work after adjusting for differences in cell density due to different numbers and yields of cells from different regions.
    3. Add ~4 ml of pre-warmed plating medium-3 to the final cell suspension (~1 ml).
    4. Transfer the cell suspension into the coated T25 culture flask, using the cotton-plugged, fire-polished Pasteur pipette. Place the flask in the culture incubator (5% CO2-95% O2, 37°C).
    5. At 2-3 DIV, when cells are 20-40% confluent, remove non-adherent or weakly adherent cells, by closing the lid tightly, shaking the flask vigorously ~10 times, aspirating the solution, and adding 4-5 ml of plating medium-3 (at 4 °C). Repeat this procedure once. Examine the cultured cells across the entire flask floor (e.g., using phase-contrast microscope) to confirm that those that appear neuronal (i.e., those for which the outer rim of the cell body appears phase-bright) are completely removed. Repeat the procedure as often as necessary to remove these cells, and then return the flask to the incubator.
      NOTE: The strength and the total number of 'shakes' required may differ among individual experimenters. The important thing is not to keep the total number of shakes to a set number, but to confirm that neuron-like cells are eliminated.
    6. At 6-8 DIV, when cells are 90-100% confluent, passage the glial cells by trypsinizing them as in step 3.2.8.
    7. After centrifugation, resuspend the pellet in 1 ml of plating medium-3 (4 °C), add 10 ml of plating medium-3 (4 °C), transfer the trypsinized cells to an un-coated T75 flask, and culture them in the incubator.
      NOTE: Rat glial cells will be passaged twice; in contrast the mouse glial cells are passaged only once because they become unhealthy after multiple passages. Rat glial cells will be passaged in T75 flasks that are not coated with any coating material. The coating allows rat glial cells to grow too quickly and yield many ciliated cells (putative ependymal cells), which will deteriorate the neuronal culture condition later.
    8. At 17-19 DIV of glial culture in a flask (i.e., 11 days after passaging and one day before trypsinization and plating onto coverslips by steps 3.3.9-3.3.14), place 100 µl of the coating material on unwashed glass coverslips (round, 12 mm diameter) in a culture dish, and place the culture dish in the culture incubator.
      NOTE: For rat glial cultures, a difference was not noticed in the culture results with or without washing the coverslips.
    9. At 18-20 DIV of glial culture in a flask (i.e., 12 days after passaging), prepare the glial feeder layer by trypsinizing the cultured cells, according to step 3.2.8.
    10. During centrifugation, aspirate the coating material completely from the glass coverslips.
    11. After centrifugation, resuspend the pellet in 1 ml of plating medium-3 (4 °C), and measure the cell density, according to step 3.1.13.
    12. Adjust the glial density to 104 live cells/ml, and plate 100 µl of the glial cell suspension on the coated glass coverslips.
      NOTE: These cells will establish the glial feeder layer.
    13. Place the culture dish with coverslips into the incubator for 20-60 min.
    14. Add 1 ml of plating medium-3 (4 °C) to each well, and place the dish back in the incubator.
    15. At 3-4 DIV of the glial feeder layer, when the cells on the coverslip are 40-80% confluent, add 1 ml of the pre-warmed growth medium (see Table 1 for composition) that contains cytosine β-D-arabinofuranoside (AraC, final concentration of 4 µM) to stop glial-cell proliferation. Plate neurons at 7-9 DIV of the glial feeder layer when the cells are 60-80% confluent, using step 3.4.
  4. Mouse Neuronal Cultures
    1. Label and genotype newborn mice according to steps 1 and 2.
    2. Use the mouse pups for step 3.1.
    3. Adjust the density of live-cell suspension to ~2.0 x 105 cells/ml using pre-warmed plating medium-2 for plating on mouse glial cells, or using plating medium-3 for plating on rat glial cells.
    4. Plate the cells on the glial feeder layer, by gently adding the cell suspension to the culture medium of each well. Note: The volume of the cell suspension to be added will be determined by the target number of cells in a culture well. For example, plate ~60 µl of cell suspension to achieve 12,000 cells/well.
    5. Note that no solution changes are necessary after the neurons are plated. Be careful to avoid evaporation of the solution from the wells over the course of culture, by humidifying the inside of the culture incubator.

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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 of age ('32-week-...

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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...

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Disclosures

The author (Zhengmin Huang) is the president of EZ BioResearch LLC that produces reagents described in this article.

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.).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
REAGENTS - tattooing
Machine CleanserAnimal Identification and Marking Systems, Inc.NMCR3This is used to clean the needles and the holder after tattooing.
Machine Drying AgentAnimal Identification and Marking Systems, Inc.NDAR4This is used to dry the needles and holder after cleaning.
Neonate Tattoo Black PigmentAnimal Identification and Marking Systems, Inc.NBP01
Skin Prep ApplicatorAnimal Identification and Marking Systems, Inc.NSPA1Q-tip.
Skin Prep solutionAnimal Identification and Marking Systems, Inc.NSP01This 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 LLCG2001-100
2X PCR Ready Mix IIEZ BioResearch LLCG2001-100A red, loading dye for electrophoresis is included in the 2X PCR Ready Mix solution.
Tissue Lysis Solution AEZ BioResearch LLCG2001-100Prepare 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 BEZ BioResearch LLCG2001-100Prepare DNA Extraction Solution by mixing 20 µl of Tissue Lysis Solution A and 180 µl of Tissue Lysis Solution B per specimen.
Acetic acid, glacialVWRBDH 3092
Agarose optimized grade, molecular biology graderpiA20090-500 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 bromideSigma-AldrichE7637-1G
Ethylenediamine tetraacetic acid, disodium salt dihydrate (EDTA)FisherBP120-500
Filtered Pipet Tips, Aerosol-Free, 0.1-10 µlDot Scientific IncUG104-96RS 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 µlDot Scientific IncUG2020-RSUse 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 µlDot Scientific IncUG2812-RSUse 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 bpEZ BioResearch LLCL1001We use either of these three molecular weight markers.
Molecular weight marker, EZ DNA Even Ladders 1000 bpEZ BioResearch LLCL1010
Molecular weight marker, TrackIt, 100 bp DNA LadderGIBCO-Invitrogen10488-058
PCR tubes, 8-tube strips with individually attached dome top caps, natural, 0.2 ml USA Scientific1402-2900Use 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 rpi145660Use tubes that are sterile and free of DNA, RNase and DNase.
Seal-Rite 0.5 ml microcentrifuge tube, naturalUSA Scientific1605-0000Use tubes that are sterile and free of DNA, RNase and DNase.
SYBR Safe DNA gel stain * 10,000x concentration in DMSOGIBCO-InvitrogenS33102
Tris baserpiT60040-1000
Primers for amplifying Tor1a gene in ΔE-torsinA knock-in mice5'-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 mice5'-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′-deoxyuridineSigma-AldrichF0503-100MGSee comments section of uridine for more information.
B-27 supplementGIBCO-Invitrogen17504-044
Cell Culture Dishes 35 x 10 mm Dishes, Tissue Culture-treatedBD falcon353001
Cell Culture Flasks, T25, Tissue Culture-treated, Canted-neck, plug-seal cap, 25 cm2 Growth Area, 70 mlBD falcon353082
Cell Culture Flasks, T75, Tissue Culture-treated, Canted-neck, vented cap, 75 cm2 Growth Area, 250 mlBD falcon353136
Conical Tube, polypropylene, 15 mlBD falcon352095
Countess (cell number counter) chamber slidesGIBCO-InvitrogenC10312
Cytosine β-D-Arabinofuranoside hydrochloride (Ara-C hydrochloride)Sigma-AldrichC6645-100mg
D-(+)-Glucose (Dextrose) anhydrous, SigmaUltra, 99.5% (GC)Sigma-AldrichG7528-250G
Dish, Petri glass 100 x 15 mmPyrex3160-101
Distilled waterGIBCO-Invitrogen15230-147
DNase Type IISigma-AldrichD4527-200KUStock 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, pyruvateGIBCO-Invitrogen10569-010, 500 ml
Fast PES Filter Unit, 250 ml, 50 mm diameter membrane, 0.2 µm Pore SizeNalgene568-0020
Fast PES Filter Unit, 500 ml, 90 mm diameter membrane, 0.2 µm Pore SizeNalgene569-0020
Fetal bovine serum (FBS)GIBCO-Invitrogen26140-079
Glass coverslip, 12 mm Round, thickness 0.09–0.12 mm, No. 0Carolina633017
GlutaMAX-IGIBCO-Invitrogen35050-061
Hanks' Balanced SaltsSigma-AldrichH2387-10X
HEPES, ≥99.5% (titration)Sigma-AldrichH3375-250G
Hydrochloric acid, 37%, A.C.S reagentSigma-Aldrich258148-100 ML
InsulinSigma-AldrichI5500-250 mg
Magnesium sulfate heptahydrate, MgSO4•(7H2O), BioUltra, ≥99.5% (Fluka)Sigma-Aldrich63138-250G
Matrigel Basement Membrane Matrix solution, Phenol Red-FreeBD Biosciences356237This 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-Invitrogen51200-038
MITO+ Serum Extender, 5 mlBD Biosciences355006
Multiwell Plates, Tissue Culture-treated 24-well plateBD falcon353047
Multiwell Plates, Tissue Culture-treated 6-well plateBD falcon353046
Neurobasal-A Medium (1X), liquidGIBCO-Invitrogen10888-022
Nitric AcidVWRbdh 3044
NS (Neuronal Supplement) 21 prepared in the labSource: reference 69
Pasteur pipets, 5 ¾” Fisher13-678-6AUse 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” Fisher13-678-6B
Potassium chloride (KCl), SigmaUltra, ≥99.0%Sigma-AldrichP9333-500G
Serological pipet, 2 mlBD falcon357507
Serological pipet, 5 ml BD falcon357543
Serological pipet, 10 mlBD falcon357551
Serological pipet, 25 mlBD falcon357525
Serological pipet, 50 ml BD falcon357550
Sodium bicarbonate (NaHCO3, Sodium hydrogen carbonate), SigmaUltra, ≥99.5%Sigma-AldrichS6297-250G
Sodium chloride (NaCl), SigmaUltra, ≥99.5%Sigma-AldrichS7653-250G
Sodium hydroxide (NaOH), pellets, 99.998% trace metals basisSigma-Aldrich480878-250G
Sodium phosphate dibasic heptahydrate (Na2HPO4•(7H2O)), ≥99.99%, AldrichSigma-Aldrich431478-250G
Sucrose, SigmaUltra, ≥99.5% (GC)Sigma-AldrichS7903-250G
Syringe filter, sterile, 0.2 µmCorning431219
Syringe, 3 mlBD falcon309585
Transferrin, Holo, bovine plasmaCalbiochem616420
Trypan Blue stain, 0.4%GIBCO-InvitrogenT10282This 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 XISigma-AldrichT1005-5G
Trypsin-EDTA solution, 0.25% GIBCO-Invitrogen25200-056
UridineSigma-AldrichU3003-5GStock 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-MAP2Merck MilliporeAB5622
Antibody, mouse monoclonal anti-GFAP cocktailMerck MilliporeNE1015
EQUIPMENT - tattooing
AIMSAnimal Identification and Marking Systems, Inc.NEO–9This 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 GTBIO–RAD170–4405Typical electrophoresis parameters are electrical field strength at 6 V/cm and 25 min duration for a 10 cm gel.
FluorChem 8800ProteinSimpleFluorChem 8800
PCR, MJ Mini Thermal CyclerBIO-RADPTC-1148EDUOur 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 BasicBIO-RAD164-5050
EQUIPMENT - cell culture
Automated cell counter, CountessGIBCO-InvitrogenC10310This 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 A2NUAIRENU-425-400This hood is used for all cell culture procedures, except for brain dissection.
CO2 Incubator, AutoFlow, Humidity Control Water JacketNUAIRENU-4850
Horizontal Clean BenchNUAIRENU-201-330This 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 ShakerLabnetS2030-LS-B
SORVALL RC-6 Plus Superspeed CentrifugeFisher46910 (centrifuge)/46922 (rotor)

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Primary Neuronal CulturesGlial Feeder LayerDNA ExtractionPCR AmplificationGel ElectrophoresisBrain DissectionTissue DigestionCell DissociationNeuronal Plating
Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons
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