Abstract
This protocol describes the technique of ex-vivo electroporation to target embryonic hippocampal progenitors in an organotypic slice preparation. This technique allows gene perturbation for examining developmental processes in the embryonic hippocampus while retaining the environment and connectivity of the cells. Gene perturbation can include Cre-mediated recombination, RNAi-mediated knockdown, gene overexpression, or a combination of any of these. Ex-vivo electroporation can be performed at a wide range of embryonic stages, giving temporal control to the experimenter. Spatial control can be achieved more easily by preparing the brain in a Petri dish to target particular regions of the hippocampus. The electroporated explant cultures provide a highly tractable system for the study of developmental processes that include progenitor proliferation, migration and cell fate acquisition.
Keywords: Mouse hippocampus, Embryo, Electroporation, Hippocampal slice, Organotypic explant
Background
The hippocampus presents a challenge in terms of accessibility due to its location in the caudomedial telencephalon. The embryonic hippocampus is even more inaccessible, requiring in utero surgical methods for experimental manipulation. Organotypic slice cultures circumvent this problem and at the same time retain many aspects of hippocampal field cytoarchitectonics, including molecular features and connectivity. While there are protocols that describe postnatal culturing of hippocampal explants from rodent brains (Stoppini et al., 1991; Opitz-Araya and Barria, 2011) these do not include genetic manipulation of the cells. The preparation of organotypic explants from the embryonic mouse hippocampus was first described in Tole et al. (1997). We extended this protocol by introducing ex-vivo electroporation of the embryonic brain prior to preparing the organotypic slices. Electroporation of the intact brain after introducing DNA into the telencephalic ventricle ensures that cells residing in and near the ventricular zone are targeted, and therefore provides an excellent means of inducing transgenesis in hippocampal progenitors. Data using this protocol were published in Subramanian et al. (2011). Here, we present detailed step-wise instructions including experimental ‘dos and don’ts’, and also illustrate key steps using photographs and movies, to aid new researchers in setting up this useful procedure.Some advantages and applications of this protocol are:1) Temporal control can be achieved by isolating the embryonic hippocampus at the desired stage to access early, mid, or late-gestation progenitors.2) Spatial control can be achieved by orienting the electrodes to target the hippocampus or different areas of the cortex. 3) Cre-mediated recombination can be employed by electroporating Cre-GFP into embryos carrying the desired floxed alleles.4) Overexpression constructs can be electroporated.5) Embryonic lethal strains can be accessed by performing the procedure in the window of viability, and then further development can proceed in the organotypic explant.
Materials and Reagents
Equipment
Procedure
Ethics statement: All procedures followed the Tata Institute of Fundamental Research Institutional Animal Ethics Committee guidelines.
Data analysis
This ex-vivo electroporated hippocampal organotypic preparation can be used to assess whether neurons or glia are generated from progenitors. The presence of GFP-expressing axons in the fimbria is indicative of neurogenesis. See Subramanian et al. (2011), Figure 5, for images of explants electroporated with neurogenic or gliogenic constructs. These explants can also be immunostained for cell type-specific markers and analyzed for the distribution of excitatory/inhibitory neurons or glia as required.
Notes
Recipes
Acknowledgments
We thank Leora D’Souza for assistance with the videography, Dr. S. Suryavanshi and the TIFR animal breeding facility for excellent support. This protocol was developed in the Tole lab using support from a Wellcome Trust Senior Fellowship (056684/Z/99/Z), a Swarnajayanti Fellowship (4/3/2005-SF), grants from the Department of Biotechnology, Government of India and intramural funds from TIFR-DAE to ST; a Kanwal Rekhi Career Development Award from the TIFR Endowment Fund to LS. ST is a recipient of the Shanti Swarup Bhatnagar award (Council of Scientific and Industrial Research, Government of India) and the Infosys Prize for Life Sciences (Infosys Science Foundation). This protocol was used in Subramanian et al. (2011) Proc Natl Acad Sci U S A 108(27): E265-274. (Current address: Eli & Edythe Broad Center of Regeneration Medicine & Stem Cell Research, University of California San Francisco, CA 94143, USA.) The authors have no conflicts of interest or competing interests.
References
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