Neonatal Subventricular Zone Electroporation

Feliciano, David M.; Lafourcade, Carlos; Bordey, Angélique

doi:10.3791/50197

Cited by 24 publications

(24 citation statements)

References 13 publications

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“…piggyBac) to mitigate this dilution problem altogether by allowing for stable insertion of transposons into the genomic DNA 29,30 . Alternatively, Cre Recombinase-expressing plasmids could be electroporated into Cre reporter mice to mediate stable genetic tracing of these cells 31 .…”

Section: Representative Resultsmentioning

confidence: 99%

“…However, as recently detailed by Jaubodin and colleagues, the targeting of postmitotic cells in this region might be possible by employing trans-cyclohexane-1,2-diol, which allows for nuclear plasmid access by permeablilizing the nuclear envelope 25 . Also, electroporation is very much compatible with Cre technologies or other engineered mouse types 31 . Finally, transposon technology will allow for stable somatic transgenesis of this population 29,30 .…”

Section: Discussionmentioning

confidence: 99%

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Neonatal Pial Surface Electroporation

Levy

Molina

Danielpour

et al. 2014

JoVE

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Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuroand gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells. Video LinkThe video component of this article can be found at

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Section: Representative Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Neonatal Pial Surface Electroporation

Levy

Molina

Danielpour

et al. 2014

JoVE

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show abstract

“…Indeed, we strongly recommend using postnatal day 2 mouse pups, since we observed a substantial decrease in labeling efficiency at later times, when viral delivery methods become more suitable 31 . However, strategies like electroporation of CRE-expressing plasmids in appropriate mouse genetic models may be used to study neurogenesis in adult stages 22 . Two-photon, standard confocal, spinning disk confocal, and wide-field fluorescence microscopy can all be used to visualize neuroblast migration 17,23,24 .…”

Section: Discussionmentioning

confidence: 99%

“…For DNA incorporation into the rostral SVZ, place electrodes slightly rostral to the injection point. Varying electrode position can achieve regional specificity of electroporation in different areas of the SVZ 22,25 . 9.…”

Section: Electroporationmentioning

confidence: 99%

<em>In vivo</em> Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Sonego

Zhou

Oudin

et al. 2013

JoVE

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The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair. Video LinkThe video component of this article can be found at

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“…This experimental procedure provides an initial, fast and relatively simple method to evaluate the role of candidate regulators of neuroblast migration, which can be further validated by other approaches like in vivo postnatal electroporation and timelapse imaging of brain slice cultures 28,31,32 .…”

Section: Prepare the Fixing Solutionmentioning

confidence: 99%

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration <em>In vitro</em>

Falenta

Gajendra

Sonego

et al. 2013

JoVE

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The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in 'hanging drops' and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

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Neonatal Subventricular Zone Electroporation

Cited by 24 publications

References 13 publications

Neonatal Pial Surface Electroporation

Neonatal Pial Surface Electroporation

<em>In vivo</em> Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration <em>In vitro</em>

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