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
Equipment
Procedure
A. Preparation of micropipettes for DNA injection
B. DNA preparation
C. Electroporation
Recipes
Acknowledgments
This protocol is adapted from Yang et al. (2004) and Ito-Ishida et al. (2012).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
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