Name: Neonatal Subventricular Zone Electroporation (Video) | JoVE Journal
Uploaded: 2013-02-11T13:00:00.000+00:00
Duration: 8 min 6 s
Description: Learn neonatal subventricular zone electroporation for genetic manipulation of neural stem cells using plasmid DNA injection and electrical pulses. This method enables rapid labeling to study neurogenesis and cell behavior.

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This work was supported by grants from the Department of Defense (Idea development award, W81XWH-10-1-0041, A.B), CT Stem cell grant (A.B.), and a National Institute of Health NRSA 10668225 (D.M.F). The present material is based on work partly supported by the State of Connecticut under the Connecticut Stem Cell Research Grants Program. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the State of Connecticut, the Department of Public Health of the State of Connecticut or CT Innovations, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This procedure is in accordance with Yale IACUC requirements. Scientists should make sure that IACUC guidelines are approved and followed according to their institutional requirements.
1. Section 1. Preparation of DNA, Solutions, and Glass Pipettes
<ol>
 <li>Generate high purity (OD 260/280 &gt; 1.80) high concentration (&mu;g/&mu;l &gt; 2.5) endotoxin-free DNA.</li>
 <li>Prepare 0.9% saline solution and sterile filter through a 22 &mu;m filter.</li>
 <li>Place 10 cm fire polished borosilicate glass capillary tubes (O.D.: 1.5 mm, I.D.:1.10 mm) into a Model PP-830 Narishige PC-10 glass pipette puller with a kanthal wire. Set puller to a one step weighted pull at 70.5 &deg;C. Break tips with circular forceps and inspect under a dissecting microscope for jagged edges. Bevel tips and place under a UV lamp for 15 min. Pulled pipettes should be marked at 2 mm from the edge of the tip.</li>
 <li>Prepare 0.1% weight/volume fast green solution by mixing sterile filtered 0.9% saline solution with dry fast green.</li>
</ol>
2. Section 2. Animal Preparation, Glass Pipette Loading and Plasmid Injection
<ol>
 <li>Remove postnatal day 0-1 pups individually from cages, place onto a glass Petri dish pre-chilled to 4 &deg;C, and then place on wet ice.</li>
 <li>After approximately 5 min, determine the state of anesthetization by utilizing the foot pinch response. If no movement occurs, subject pups to intraventricular injection.</li>
 <li>It is important to note that the combination of fast green, DNA, and saline solution can result in rapid evaporation, crystallization, and blockade of glass pipettes. Therefore, generate a stock solution and aliquot 1-2 &mu;l onto parafilm immediately prior to injections.</li>
 <li>Aspirate the entire aliquoted volume with the pulled glass pipette.</li>
 <li>Hold the head of the pup between your thumb and index finger of your less dominant hand.</li>
 <li>Turn on lamp and hold head of pup just outside of light beam. If needed, put saline on head to reduce the amount of light reflected by pup. The ventricles should now be illuminated.</li>
 <li>With your dominant hand, insert the needle into lateral ventricle closest to you (Figure 1A and 1B). The site of injection should be approximately equidistant from the lambdoid suture and eye and 2 mm lateral to the sagittal suture. Insert pulled pipette to the 2 mm mark for a P0 pup, which should ensure penetration into the lateral ventricle. Coincidentally, you may see back filling of CSF into the pipette from the release of intracranial pressure. To reduce intracranial pressure, which assists in injection of plasmid, loosen your grip on the head of the pup.Step 2.8. Inject approximately 0.5 &mu;l of plasmid into lateral ventricle using an air-pressured injector (Picospritzer, Parker).</li>
</ol>
3. Section 3. Electroporation and Recovery
<ol>
 <li>Set the voltage of the electroporation generator for 5 square pulses, 50 msec/pulse at 100 volts, with 950 msec intervals. The spatial specificity of plasmid electroporation is dictated by the directionality of charge transfer. Close attention should be given to the placement given the heterogeneity of stem cell populations along the SVZ8. Differences in the rates of proliferation and cell fate have been identified for ventral-dorsal and rostral-caudal locations9,10 .</li>
 <li>Once the plasmid is injected, dip tweezer electrodes into PBS. This step is to maximize charge transfer and to prevent burns caused by resistivity.</li>
 <li>Place the positive electrode on the dorsal lateral wall of the pup near the ear ipsilateral to the site of plasmid injection (Figure 1C and 1D). Place the negative electrode on the contralateral hemisphere ventral lateral to the pup's snout.</li>
 <li>Initiate current transfer by pressing the pulse footswitch pedal and sweep the electrodes from dorsal to lateral using ~25 &deg; angle intervals.</li>
 <li>Place electroporated pups onto a heating pad for 5 min. Gently rotate pups every 30 sec with mild stimulation.</li>
</ol>

<ol><li>Kriegstein, A., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+glial+nature+of+embryonic+and+adult+neural+stem+cells%5BTitle%5D)+AND+%22Annual+Review+of+Neuroscience%22%5BJournal%5D)">The glial nature of embryonic and adult neural stem cells.</a> Annual Review of Neuroscience. 32, 149-184 (2009).</li>
<li>Imayoshi, I., Sakamoto, M., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+methods+to+identify+and+manipulate+newly+born+neurons+in+the+adult+brain%5BTitle%5D)+AND+%22Frontiers+in+Neuroscience%22%5BJournal%5D)">Genetic methods to identify and manipulate newly born neurons in the adult brain.</a> Frontiers in Neuroscience. 5, 64(2011).</li>
<li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex%5BTitle%5D)+AND+%22Cereb+Cortex%22%5BJournal%5D)">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cereb Cortex. 19, Suppl 1. i120-i125 (2009).</li>
<li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation%3A+visualization+of+neuronal+migration+in+the+developing+cortex%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li>
<li>Boutin, C., Diestel, S., Desoeuvre, A., Tiveron, M. C., Cremer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+in+vivo+electroporation+of+the+postnatal+rodent+forebrain%5BTitle%5D)+AND+%22PloS+ONE%22%5BJournal%5D)">Efficient in vivo electroporation of the postnatal rodent forebrain.</a> PloS ONE. 3, e1883(2008).</li>
<li>Chesler, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+gene+expression+by+postnatal+electroporation+during+olfactory+interneuron+nurogenesis%5BTitle%5D)+AND+%22PloS+ONE%22%5BJournal%5D)">Selective gene expression by postnatal electroporation during olfactory interneuron nurogenesis.</a> PloS ONE. 3, e1517(2008).</li>
<li>Platel, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NMDA+receptors+activated+by+subventricular+zone+astrocytic+glutamate+are+critical+for+neuroblast+survival+prior+to+entering+a+synaptic+network%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">NMDA receptors activated by subventricular zone astrocytic glutamate are critical for neuroblast survival prior to entering a synaptic network.</a> Neuron. 65, 859-872 (2010).</li>
<li>Alvarez-Buylla, A., Kohwi, M., Nguyen, T. M., Merkle, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+heterogeneity+of+adult+neural+stem+cells+and+the+emerging+complexity+of+their+niche%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Symposia+on+Quantitative+Biology%22%5BJournal%5D)">The heterogeneity of adult neural stem cells and the emerging complexity of their niche.</a> Cold Spring Harbor Symposia on Quantitative Biology. 73, 357-365 (2008).</li>
<li>Fernandez, M. E., Croce, S., Boutin, C., Cremer, H., Raineteau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+electroporation+of+defined+lateral+ventricular+walls%3A+a+novel+and+rapid+method+to+study+fate+specification+during+postnatal+forebrain+neurogenesis%5BTitle%5D)+AND+%22Neural+Development%22%5BJournal%5D)">Targeted electroporation of defined lateral ventricular walls: a novel and rapid method to study fate specification during postnatal forebrain neurogenesis.</a> Neural Development. 6, 13(2011).</li>
<li>de Chevigny, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miR-7a+regulation+of+Pax6+controls+spatial+origin+of+forebrain+dopaminergic+neurons%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">miR-7a regulation of Pax6 controls spatial origin of forebrain dopaminergic neurons.</a> Nature Neuroscience. 15, 1120-1126 (2012).</li>
<li>Lacar, B., Young, S. Z., Platel, J. C., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+and+recording+subventricular+zone+progenitor+cells+in+live+tissue+of+postnatal+mice%5BTitle%5D)+AND+%22Frontiers+in+Neuroscience%22%5BJournal%5D)">Imaging and recording subventricular zone progenitor cells in live tissue of postnatal mice.</a> Frontiers in Neuroscience. 4, (2010).</li>
<li>Feliciano, D. M., Quon, J. L., Su, T., Taylor, M. M., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postnatal+neurogenesis+generates+heterotopias%2C+olfactory+micronodules+and+cortical+infiltration+following+single-cell+Tsc1+deletion%5BTitle%5D)+AND+%22Human+Molecular+Genetics%22%5BJournal%5D)">Postnatal neurogenesis generates heterotopias, olfactory micronodules and cortical infiltration following single-cell Tsc1 deletion.</a> Human Molecular Genetics. 21, 799-810 (2012).</li>
<li>Iguchi, T., Yagi, H., Wang, C. C., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+tightly+controlled+conditional+knockdown+system+using+the+Tol2+transposon-mediated+technique%5BTitle%5D)+AND+%22PloS+ONE%22%5BJournal%5D)">A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.</a> PloS ONE. 7, e33380(2012).</li>
</ol>

The authors have no disclosures to make.

Here we detail the technique of neonatal SVZ electroporation, a technique to rapidly and robustly label and manipulate SVZ stem cells and their progeny. There are several advantages that electroporation has in comparison to other techniques. First, given the focal labeling of cells, one is able to discern cell autonomous and non-cell autonomous effects. Second, genetic manipulation using inducible systems allows one to compare effects prior to or after synaptic integration. Furthermore, one can bypass the use of m...

<table><tbody><tr><td>Heavy Polished Borosilicate Tubing </td><td>Sutter Instrument</td><td>BF150-110-10</td><td></td></tr><tr><td>Dual-Stage Glass Micropipette Puller </td><td>Narishige</td><td>PC-10H</td><td></td></tr><tr><td>Fast Green </td><td>Fischer Scientific</td><td>0521192205</td><td></td></tr><tr><td>ECM 830 Square Wave Pulse generator </td><td>Harvard Apparatus</td><td>45-0052</td><td></td></tr><tr><td>Tweezertrodes</td><td>Harvard Apparatus</td><td>45-0488</td><td></td></tr><tr><td>Fiber-Optic Light Source</td><td>Fisher Scientific</td><td>12-562-36</td><td></td></tr><tr><td>Tungsten Halogen lamp</td><td>USHIO America, Inc</td><td>1002247</td><td></td></tr><tr><td>Picospritzer II</td><td>Parker Instruments</td><td>052-0312-900</td><td></td></tr></tbody></table>

neonatal subventricular zone electroporation

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

Neonatal subventricular zone electroporation results in the labeling of nearly all radial glia contiguous with the dorsal, dorsal lateral, and lateral subventricular zone following the "sweeping" movement of the tweezer electrode (Figure 1E). However, electroporation can be tailored to the requirement of the respective experiment but not sweeping the electrode and using specific placement and orientation as detailed in the video. For example, since dorsally localized radial glia differ from those ...

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

The aim of this procedure is to genetically label and manipulate the molecular constituents of sub ventricular zone neural stem cells. This is accomplished by first inserting a glass pipette loaded with DNA into the lateral ventricle of a P zero P.The second step is to expel DNA into the cerebral spinal fluid of the lateral ventricle. Next current is applied to the P.This allows for transfer of DNA to neural stem cells lining the lateral ventricles.Ultimately, a combination of molecular techniques, including immunohistochemistry, molecular biology, microscopy, and electrophysiology, are used to visualize changes in fate, proliferation, migration, and maturation of subventricular zone neuro stem cells. Neonatal subventricular zone electroporation can help answer key questions in the field of neurogenesis, such as which molecular pathways are responsible for regulating the proliferation, differentiation, migration, and maturation of neuronal stem cells and their progeny. The main advantage of this technique over existing methods, such as transgenic mice, is that it allows for the rapid and robust labeling of neuron stem cells of the SVZ in a time efficient and cost-effective manner.First, ensure that the DNA to electro purate is of high purity and concentration and is endotoxin free. Pull 10 centimeter fire polished BO silica glass capillary tubes into pipettes. A PP eight 30 ghhi PC 10 pipette puller set to pull a one step weighted pull at 70.5 degrees Celsius is used here After pulling the pipette, use circular forceps to break the tip.Then inspect the pipette under a dissecting microscope to ensure that there are no jagged edges. If required, the tip can be beveled using a grindstone. Next place the poured pipettes under a UV lamp for 15 minutes.Once the 15 minutes have elapsed, mark the pipette two millimeters from the edge of the tip with a fine tip marker. Finally, prepare a 0.1 weight per volume fast green solution by mixing sterile filtered 0.9%sterile saline with dry, fast green anesthetize. A zero to one day old pup, a glass Petri dish pre chilled to four degrees Celsius and placed on west ice After approximately five minutes, determine the state of anesthetization by utilizing the foot pinch response.If no movement occurs, begin the intraventricular injection. Combine the fast green solution with the DNA solution and aliquot one to two microliters onto biofilm. It is important that this is done just prior to injection as the combination of fast green solution with DNA can result in rapid evaporation and crystallization with the subsequent blocking of glass pipettes, aspirate the entire aliquot volume with a microfilm syringe and load the glass pipette onto an air pressured injector.Now, gently grasp and hold the head of the pup between the thumb and forefinger of the last dominant hand. Then turn on the lamp and hold the head of the pup just outside of the light beam. If the ventricles are not clearly visible, dampen the head with sterile saline to help reduce the amount of light reflected by the scalp.Next, using the dominant hand, insert the needle into the near side lateral ventricle. The site of injection should be approximately equidistant from the OID suture and eye, and two millimeters lateral to the sagittal suture. Insert the pulled pipette to the two millimeter mark for a P zero pop, which should ensure penetration into the lateral ventricle.Back filling of CSF into the pipette may occur due to the release of intracranial pressure. If this happens, loosen the grip on the head of the POP to reduce the pressure and assist in the injection of the plasmid. Once the pipette is optimally inserted, inject approximately 0.5 microliters of plasmid into the lateral ventricle.Set the voltage of the electro operation generator for five square pulses, 50 milliseconds per pulse, and 100 volts with 50 millisecond intervals. The spatial specificity of plasma electro operation is given by the direction of the charge transfer, so spatial consideration should be given to the placement of the electrodes given how diverse the cells of the SVZ are. People have reported changes in terms of cell fate and proliferation in the ventral dorsal to CAU locations.After injection of the plasmid dip, the tweezer electrodes into PBS to maximize charge transfer and help prevent burns caused by resistivity. Next, place the positive electrode on the dorsal lateral wall of the skull near the ear and dips the lateral to the cytoplasm injection. Then place the negative electrode on the contralateral hemisphere ventral lateral to the pop snout.This figure shows the correct orientation of negative and positive electrodes with respect to the x, y, Z axis on the injected pup. Initiate current transfer by pressing the pulse foot switch pedal and sweep the electrodes from dorsal to lateral using around 25 degree angle intervals. Once the electroporation is complete, place all electroporated pups onto a heating pad for five minutes.Gently rotate the pups every 30 seconds with mild stimulation. This image shows the lateral ventricle of a neonatal mouse 24 hours following electroporation of an EGFP encoding vector. The electro rated cells fluoresce green.Here we see the lateral ventricle labeled LV 28 days after electroporation of Cree Recombinase and GFP encoding plasmids into mice carrying a stop code on upstream of TD tomato recombination induces expression of TD tomato seen in red, successive dilution of plasmid resulting few remaining EGFP positive SVZ cells or genomically expressed TD tomato is permanently expressed. Dilution of plasmid, DNA shown in green is apparent given that more cells in the granule cell layer of the olfactory bulb labeled OB, expressed the genomic marker TD tomato shown in red. After watching this video, you should have a good understanding of how to label and manipulate stem cells from the SVZ.

neonatal-subventricular-zone-electroporation

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Neuroscience

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

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention.  Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method.  This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

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In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

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The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

Learn neonatal subventricular zone electroporation for genetic manipulation of neural stem cells using plasmid DNA injection and electrical pulses. This method enables rapid labeling to study neurogenesis and cell behavior.

Neonatal Subventricular Zone Electroporation

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