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This protocol will be useful to introduce the genes of interest into the cerebellar granule cells at early stages of development. Since the granule cell precursors are localized in the external granule layer before migration, DNA plasmids can be specifically incorporated into the granule cells by injecting DNA solution into the cerebellar fissures followed by application of electric pulses. This technique can be performed prior to the preparation of either dissociated or organotypic culture, which can be used to study the molecular mechanisms of cell migration, axon elongation and synapstogenesis during development.

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Labeling of Precursor Granule Cells in the Cerebellum by ex vivo Electroporation

Developmental Biology > Cell growth and fate > Neuron
Author: Aya Ito-Ishida
Aya Ito-IshidaAffiliation: Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
For correspondence: barquebleue@hotmail.com
Bio-protocol author page: a385
Vol 3, Iss 12, 6/20/2013, 4415 views, 1 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.778

[Abstract] This protocol will be useful to introduce the genes of interest into the cerebellar granule cells at early stages of development. Since the granule cell precursors are localized in the external granule layer before migration, DNA plasmids can be specifically incorporated into the granule cells by injecting DNA solution into the cerebellar fissures followed by application of electric pulses. This technique can be performed prior to the preparation of either dissociated or organotypic culture, which can be used to study the molecular mechanisms of cell migration, axon elongation and synapstogenesis during development.

Keywords: Electroporation, Cerebellum, Granule cell, Neuron

Materials and Reagents

  1. Plasmid Maxi Kit (QIAGEN)
  2. 10x HEPES buffered saline (HBS)
  3. Fast green FCF (Sigma-Aldrich, catalog number: F7252)
  4. 1x Phosphate buffered saline (PBS)
  5. 10x HBS (see Recipes)

Equipment

  1. Capillary glass (Harvard Apparatus, catalog number: 30-0066)
  2. Micropipette puller P-97/ IVF (Sutter Instrument)
  3. Aspirator Tube Assembly (Drummond, catalog number: 2-000-000)
  4. Square pulse electroporator and a foot switch (Nepagene, catalog number: CUY21)
  5. Platinum plate tweezers-type electrode (Protech International, catalog number: CUY650-P5)
  6. Dissecting microscope
  7. Dissecting tools: Forceps and some fine scissors
  8. 10 cm-petri dish

Procedure

A.   Preparation of micropipettes for DNA injection

  1. Pull the capillary glass using the puller with a single pull. Adjust the puller setting to make the capillary taper approximately 10-20 mm. When using P-97, start with the following setting: Heat equal of the Ramp value, Pull 0, Vel 40 and Time 200.
  2. Cut out 1/3 – 1/2 of the tips of the pulled pipettes using the forceps.

B.  DNA preparation

  1. Prepare plasmids using the Plasmid Maxi Kit and elute DNA with distilled water. The concentration of the DNA should be 4-5 μg/μl for storage.
  2. For injection, the final concentration of DNA should be 2 μg/μl. Add 1/10 volume of 10x HBS, 1/10 volume 1% fast green (final 0.1%) and water to the DNA solution to adjust the concentration.

C. Electroporation

  1. Attach a micropipette to an aspirator tube assembly and draw 10 μl of DNA -solution into the pipette (Figure 1).


    Figure 1. An aspirator tube and a glass capillary

  2. Remove a whole-brain from a mouse (postnatal days 7-9) and put it into an ice-cold PBS in a 10-cm petri dish.
  3. Remove the forebrain and the brain stem using the forceps. If the cerebellum is planned to be used for organotypic culture, the meninges should not be removed in order to keep the cellular layers intact. If the cerebellum is to be further processed for dissociated culture, the meninges should be removed to avoid glial cell overgrowth.
  4. Observe the cerebellum under a dissecting microscope. Inject the DNA solution (2 -3 μl/fissure) into the cerebellar fissures by mouth pipetting (Figure 2). Inject into at least 2 or 3 fissures, starting from those in the vermis. If high transfection efficiency is required, inject into as many fissures as possible.


    Figure 2. Injection of plasmid DNA solution into cerebellar fissures

  5. Place the forceps-type electrodes so that cerebellum is in between the two electrode planes. The two planes should be positioned parallel to the fissures of the cerebellar vermis. They should be held about 3-5 mm apart from the cerebellum surface (Figure 3).


    Figure 3. Placement of electrodes

  6. Apply electric pulses; 99.9 V, ON 50 msec, OFF 450 msec, 5 pulses. Adjust the voltage value depending on the outcome. When the transfection efficiency is too low, the voltage should be increased up 120 V. When the transfection efficacy is too high or when the number of dying cells is too high, the voltage setting should be decreased.
  7. The cerebellum can be further processed for either organotypic slice or dissociated cultures (please see reference 1 for the images showing the labeled granule cells).

Recipes

  1. 10x HBS

    10x conc. (mM)
    g/100 ml H2O
    NaCl
    1,400
    8.2
    Na2HPO4.2H2O
    15
    0.3
    HEPES
    500
    11.9

Acknowledgments

This protocol is adapted from Yang et al. (2004) and Ito-Ishida et al. (2012).

References

  1. Ito-Ishida, A., Miyazaki, T., Miura, E., Matsuda, K., Watanabe, M., Yuzaki, M. and Okabe, S. (2012). Presynaptically released Cbln1 induces dynamic axonal structural changes by interacting with GluD2 during cerebellar synapse formation. Neuron 76(3): 549-564.
  2. Yang, Z. J., Appleby, V. J., Coyle, B., Chan, W. I., Tahmaseb, M., Wigmore, P. M. and Scotting, P. J. (2004). Novel strategy to study gene expression and function in developing cerebellar granule cells. J Neurosci Methods 132(2): 149-160.


How to cite this protocol: Ito-Ishida, A. (2013). Labeling of Precursor Granule Cells in the Cerebellum by ex vivo Electroporation. Bio-protocol 3(12): e778. DOI: 10.21769/BioProtoc.778; Full Text



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2/7/2014 10:04:27 AM  

Mariana Silveira
Instituto de Biofísica Carlos Chagas Filho, UFRJ.

Dear Dr Ito-Ishida,
I am interested in obtaining details on your ex vivo electroporation protocol for cerebellar progenitors.
I would really appreciate if you could answer some of my doubts. Please feel free to send me any other detail you consider relevant.
- we had a hard time to build the capillary device you use. Have you ever tried to use Hamilton syringe? Do you believe this could work?
- How do you restrict the diffusion to occur after the use of the capillary?
Sorry to bother you and thank you in advance for your help.
Regards,
Mariana Silveira
silveira@biof.ufrj.br

2/7/2014 8:29:14 PM  

Aya Ito-Ishida (Author)
Department of Cellular Neurobiology, Graduate School of Medicine,University of Tokyo

Dear Dr. Silveira,
1. I have never used Hamilton syringe for this purpose myself, but I think it is possible. You may need to practice several times so that the plasmid-containing liquid will go into the fissures slowly. It may be easier to try it with another person's help: one person could hold the cerebellum and another person could manipulate the syringe.
2. When injecting the plasmid-containing liquid, the tip of the capillary has to be within the cerebellar fissures. The two cerebellar lobules will make walls on both sides of the capillarly tip and prevent the liquid from diffusing. In addition, the electric pulses should be applied right after the injection.
_ I hope this helps. Please let me know if you have any further questions.
Best wishes,
Aya Ito-Ishida

Reply

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