We acknowledge the help of M.J. Palop and the technical support of the SCSIE of the Universidad de Valencia. We also thank Antonia Follenzi for helpful comments and discussion of the manuscript. I.F is supported by Fundaci&#242;n Bot&#236;n, by Banco Santander through its Santander Universities Global Division, and by grants from Generalitat Valenciana (Programa Prometeo, ACOMP, and ISIC) and Ministerio de Econom&#236;a y Competitividad (MINECO: SAF2011-23331, CIBERNED and RETIC TerCel). This work was also supported by BFU2010-21823 and RETIC TerCel grants from MINECO and the European Research Council (ERC) 2012-StG (311736- PD-HUMMODEL) to A.C. B.M-P. is the recipient of a Spanish FPI fellowship of the MINECO.

ETHICS STATEMENT: This protocol follows the animal care guidelines of the University of Valencia in compliance with European directive 2010/63/EU.
1. Generation of LV for In Vivo Marking Studies (see Figure 1a)
CAUTION: The procedure described herein is biosafety level 2, therefore perform all the following procedures in a biohazard hood. Ensure that research personnel are appropriately qualified and trained in all procedures. Wear personal protective equipment, including gown, double gloves and suitable eye protection. Finally, thoroughly decontaminate all tools and surfaces that could have been in contact with viruses according to approved facility disinfection practices (by wiping with 70% ethanol, 10% bleach and/or autoclaving).
<ol>
	<li>Production of LV in Human Embryonic Kidney 293T Cells
	<ol>
		<li>Start this protocol by preparing pure DNA for transfection. Prepare and purify each plasmid by double CsCl gradient centrifugation or other commercially available column methods yielding endotoxin-free DNA. In this protocol we have used the transfer vector plasmid pRRL-SIN-PPT.PGK.EGFP.Wpre. Recommended core packaging plasmids are pMDLg/pRRE and pRSV.REV and envelope plasmid pMD2G13,18,19.</li>
		<li>Twenty-four hr before transfection, plate 5 x 106 293T cells in Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM) (see Table of Materials) in a 10 cm plastic dish in order to obtain an approximately 1/4 to 1/3 confluent culture for transfection. Incubate at 37 &#176;C in a humidified incubator in an atmosphere of 5-7% CO2.</li>
		<li>Replace the medium with fresh medium 2 hr before transfection.</li>
		<li>In a sterile 1.5 ml microcentrifuge tube mix 10 &#181;g of transfer vector plasmid (containing the cDNA of the transgene or the shRNA to be delivered) with 2.5 &#181;g of the pRSV.REV and 5 &#181;g of the pMDLg/pRRE packaging plasmids, and 3.5 &#181;g of the envelope plasmid pMD2G. Make up the plasmid solution to a final volume of 450 &#181;l with 0.1x TE buffer (see Table of Materials) /dH20 (2:1). Then add 50 &#181;l of 2.5 M CaCl2.</li>
		<li>Form the precipitate by dropwise addition of 500 &#181;l of the 2x Hepes Buffered Saline(HBS, see Table of Materials) solution to the 500 &#181;l DNA-TE-CaCl2 mixture while vortexing at full speed.</li>
		<li>Add the precipitate to the 293T cells immediately. Gently swirl the plate to mix. Return the cells to the incubator and change the medium 14-16 hr after transfection.</li>
		<li>Collect the cell supernatants 30 hr after changing the media. Filter supernatant through a 0.22 &#181;m pore nitrocellulose filter and proceed to concentration.</li>
	</ol>
	</li>
	<li>Concentration of LVs
	<ol>
		<li>Concentrate the conditioned medium by ultracentrifugation at 50,000 x g (19,000 rpm with SW-28 ultracentrifuge rotor) for 2 hr at room temperature (RT) in a 30 ml polypropylene transparent conical rotor tube. 
		Note: Use ultracentrifuge adapters for conical rotor tubes (see table of Materials).</li>
		<li>Discard the supernatants by decanting and resuspend the pellets in a small volume (200 &#181;l or less if only one centrifugation is performed) of phosphate buffer saline (PBS; see Table of Materials). Then pipette up and down about 20 times.</li>
		<li>Pool the suspensions and concentrate again by ultracentrifugation, also at 50,000 x g (23,000 rpm with SW-55 ultracentrifuge rotor) for 2 hr at room temperature. Use polypropylene transparent rotor tubes with a nominal volume of 5 ml (see Table of Materials).</li>
		<li>Resuspend the final pellet in a very small volume (1/500 or 1/1,000 of the starting volume of medium) of sterile PBS and shake on a rotating wheel for 1 hr at RT. Split into small aliquots (5-20 &#181;l) and freeze them at -80 &#176;C.</li>
		<li>Treat all empty tubes with 10% bleach before discarding.</li>
	</ol>
	</li>
	<li>Lentiviral Titration Using Flow Cytometry
	<ol>
		<li>The day before, plate 5 x 104 HeLa cells per well in 6-well tissue culture plates in 2 ml Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) (see Table of Materials). Incubate at 37 &#176;C in a humidified incubator in an atmosphere of 5-7% CO2 for 24 hr.</li>
		<li>On the day of titration, thaw an aliquot of the viral stock and prepare serial dilutions, from 10-3 to 10-8, in DMEM.
		<ol>
			<li>To do so, take a 24-well plate and add 2 ml of DMEM to the first well and 1.8 ml to the following wells. Then add to the first well 2 &#181;l of the concentrated viral stock (to a final dilution of 1:1,000 or 10-3).</li>
			<li>After pipetting several times to thoroughly mix the solution, change tip and transfer 200 &#181;l of the 10-3 dilution to the second well. Repeat the procedure serially in the following wells until the 10-8 dilution is made.</li>
		</ol>
		</li>
		<li>Take HeLa cells plated the previous day from the incubator. Carefully remove medium from wells. Add 1 ml of each viral dilution together with 1 &#181;l of 8 mg/ml hexadimethrine bromide to the HeLa cell-containing wells. Gently swirl the plate to mix. 
		Note: Hexadimethrine bromide is added to increase the virus adsorption to the cells in culture.</li>
		<li>Return the cells to the incubator and allow the infection to proceed for 72 hr. After that, remove the medium, wash the cells once with PBS and add 200 &#181;l of trypsin-EDTA (see Table of Materials) to each well.</li>
		<li>After 5 min at 37 &#176;C, add 2 ml of PBS to each well and harvest cells in flow cytometry tubes.</li>
		<li>Centrifuge at 300 x g for 5 min at RT and aspirate the supernatant.</li>
		<li>Resuspend the pellet with 1 ml of fixing solution (1% formaldehyde electron microscopy grade and 2% fetal bovine serum in PBS), then vortex the tubes.</li>
		<li>Analyze the cells in a flow cytometer using a 488 nm argon-ionlaser at 15 mW power.</li>
		<li>Set up the instrument with the standard configuration: forward-scatter (FS), side-scatter (SS), and fluorescence for GFP (525/40 nm). Select cell population gating in a FS vs. SS dot plot to exclude cell aggregates and debris. Collect fluorescence in logarithmic scale. Calculate the number of GFP+ cells in each sample.</li>
		<li>Calculate vector titer using the following formula: % GFP+/100 x number of cells infected x dilution factor (DF) = transducing units (TU)/ml.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53282/53282fig1.jpg" /> 
Figure 1: Schematic representation of the different parts of the procedure. (a) Part 1 of the protocol: generation of LVs for in vivo labeling studies, from the transfection of HEK293T cells with appropriate plasmids to generate the LVs to the determination of the virus titer by flow cytometry using the indicated formula. The names of the plasmids and the centrifuge rotors are indicated. (b and c) Part 2 of the protocol: stereotaxic injection of LVs. &#34;b&#34; depicts an example of a Vernier scale, a device that is part of stereotaxic instruments and serves for fine measurements. As an example, the arrows indicate 4.23 cm. A Vernier scale is used to determine the coordinates in the antero-posterior (AP), medio-lateral (ML), and dorso-ventral (DV) axis as shown for a top-view (left) and for a sagittal section (right) of the brain. &#34;c&#34; indicates the position of bregma as the intersection between the sagittal and coronal sutures. LVs are injected using a syringe. (d) Schematic drawings showing how the brain is processed for analysis. The two hemispheres are split and each one is divided into two blocks. Block &#34;a&#34;, containing the OBs, is produced by a coronal cut at the AP level immediately posterior to the OB junction with the telencephalon (bregma 2.46 mm; see Paxinos&#180; Atlas for a reference). Block &#34;b&#34; is produced by two coronal cuts, one at the level just anterior to the most rostral aspect of the corpus callosum (bregma 1.7 mm) and a second one at the level of the junction of the two lateral ventricles (bregma -0.22 mm). GL, glomerular layer; GCL, granule cell layer; st, striatum; cc, corpus callosum; ac, anterior commissure; lv, lateral ventricle.
2. Stereotaxic Injection of LV into the V-SVZ/Striatum Border or into the Lateral Ventricle (see Figure 1b)
<ol>
	<li>Preparation
	<ol>
		<li>Sterilize a 5 &#181;l capacity syringe with a 33 gauge needle by spraying down the body and needle with 70% ethanol with the plunger pulled out all the way. Repeatedly aspirate ethanol from a 1.5 ml microcentrifuge tube and eject it all the way out several times, and rinse the syringe thoroughly with sterile water afterwards. Place the syringe safely aside in the culture hood and allow it to dry.</li>
		<li>Prepare a biohazard waste container with 10% bleach to a suitable volume for immersion of all waste from this procedure (generally 200 ml in a 500 ml container).</li>
		<li>Prepare and preheat a 37 &#176;C waterbed by filling a sealable plastic storage bag with water and warming it to 37 &#176;C. This will allow mice to recover following injection.</li>
		<li>Remove viral stocks from -80 &#176;C freezer storage 1 hr before starting the injections and place the vial on a rotating wheel at RT. After thawing, maintain the viral stock on ice during the time of the injections. Prior to the stereotaxic injection of LV, dilute the concentrated viral stocks to 106 TU/&#956;l using PBS in the culture hood.</li>
		<li>Sanitize the area selected for performing the surgery with 70% ethanol.</li>
	</ol>
	</li>
	<li>Microinjection of LV
	<ol>
		<li>Select and sterilize tools needed for surgery (scalpel, drill, and small tweezers).</li>
		<li>Anesthetize a 6-8 week-old mouse by intraperitoneally (ip) injecting a veterinary-supervised mixture of ketamine and medetomidine. Weigh each animal and dose each with 50-75 mg ketamine and 0.5-1 mg medetomidine per kg of mouse body weight (around 100-125 &#181;l of the ketamine/medetomidine working solution per mouse).</li>
		<li>Assess the anesthetic plane by pinching the toes, tail or ear and ensuring that the animal shows no reaction.</li>
		<li>Once the mouse is anesthetized, inject butorphanol subcutaneously at a final dose of 0.4-0.5 mg per kg mouse weight to minimize post-surgical pain.</li>
		<li>Shave the area between the ears and disinfect the skin using an iodophor such as iodopovidone or 70% ethanol. Cleanse using sterile cotton-tipped applicators. Be careful not to excessively wet the animal as this can exacerbate hypothermia.</li>
		<li>Place the animal in prone position on a stereotaxic frame and carefully fix the head using the ear bars and the palate support of the apparatus. Keep the mouse with a heating pad set at 37 &#176;C and apply ophthalmic lubricant to the eyes.</li>
		<li>Make a 1 cm long incision on the head skin longitudinally using a scalpel, and gently retract the skin to expose the skull using fine tweezers.</li>
		<li>Carefully clean the bone surface with a sterile cotton-tipped applicator. Cleanse the exposed skull bone of any remaining tissue.</li>
		<li>Mount the sterilized syringe on the stereotactic device using the syringe holder.</li>
		<li>Move the syringe holder x, y and z axis until the tip of the syringe needle is positioned on the bregma, the conjunction point where the sagittal (longitudinal and medial) suture is perpendicularly intersected by the coronal suture (Figure 1b). Ensure that the &#34;zero&#34; position of the dorso-ventral (DV) axis is at the skull surface at bregma.</li>
		<li>Move the syringe to the x and y destination coordinates (see Table 1 and Figure 1b).</li>
	</ol>
	</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td rowspan="2">Region of injection</td>
			<td colspan="3">Coordinates</td>
		</tr>
		<tr>
			<td>Antero-posterior (AP)</td>
			<td>Medio-lateral (ML)</td>
			<td>Dorso-ventral (DV)</td>
		</tr>
		<tr>
			<td>SEZ/striatum border</td>
			<td>+0.6 mm</td>
			<td>+1.2 mm</td>
			<td>-3.0 mm</td>
		</tr>
		<tr>
			<td>Lateral ventricle</td>
			<td>-0.3 mm</td>
			<td>+1.0 mm</td>
			<td>-2.6 mm</td>
		</tr>
	</tbody>
</table>
Table 1: Stereotaxic coordinates for the injections. For the AP and ML axis, x and y coordinates are given as a distance (in mm) from bregma. &#34;-&#34; indicates &#34;towards posterior&#34;. For the DV coordinates &#34;zero&#34; is the surface of the skull at the bregma point and DV coordinates indicate the distance (in mm) down from this point.
<ol start="12" style="margin-left: 40px;">
	<li>Annotate the x, y and z destination coordinates in the Vernier scale in order to be able to come back to the injection site later on. Mark the bone at the x and y coordinates using a surgical marker pen.</li>
	<li>Move the syringe away from the working area.</li>
	<li>Using an electric drill make a hole on the skull carefully not to damage the brain. Do not drill the pial surface as this may damage the brain surface.</li>
	<li>Load the syringe with 1 &#181;l of the 106 TU/&#181;l viral solution. Use a 33 gauge sharp beveled needle whose tip has an angle of 10-12&#176;. Position the syringe needle at a 90&#176; angle with respect to the brain surface.</li>
	<li>Move the syringe back to the site of injection and move it down until the tip touches the pial surface.</li>
	<li>Penetrate the brain with the syringe to the z coordinate in the DV axis.</li>
	<li>Slowly release the viral suspension, at a rate of 0.2 &#181;l/min, in order to minimize damage to the brain tissue due to excessive fluid pressure.</li>
	<li>Wait for 5-10 min to minimize the backflow of viral suspension and then retract the syringe very slowly. Blot any excess of liquid that may appear at the surface as a result of the retraction of the syringe using a laboratory wipe and place it immediately in the bleach-containing biosafety waste container.</li>
	<li>Take the animal out of the stereotaxic set, place it on a warm pad, and close the wound using skin adhesive. Reverse the sedation using 0.1-1.0 mg/kg body weight atipamezole.</li>
	<li>Inject Bupenorphrine subcutaneously at a final dose of 0.1 mg per kg mouse weight every 12 hr, starting 4 hr after the administration of the short lasting Butorphanol analgesic.</li>
	<li>Place the animal in an individualized cage with a warm pad and monitor closely until the mouse recovers from anesthesia. Place one bag of hydrogel in the cage to help the animal hydrate after recovery.</li>
	<li>Dispose of all bio-contaminated waste in the liquid bleach biohazard disposal. Clean the syringe by aspiration and ejection of ethanol and rinse with water. Disinfect the area, the stereotaxic set and the surgical material that has been used with bleach and 70% ethanol.</li>
	<li>Keep injected mice isolated in the biosafety level 2 room for 24-48 hr after which they can be transferred to a conventional housing facility</li>
</ol>
3. Histological Analysis
<ol>
	<li>Perfusion, tissue collection, and sectioning
	<ol>
		<li>Deeply anaesthetize the mice using a veterinary-supervised mixture of medetomidine and ketamine (assess the anesthetic plane by pinching the toes, tail or ear), as described before.</li>
		<li>Transcardially perfuse the mice with 25 ml of saline solution followed by 75 ml of 4% PFA in PB at the same rate17.</li>
		<li>Extract the brain and post-fix it by immersing it in at least 10 times its volume of cold 4% PFA in PB for 1-16 hr (increased post-fixation times may decrease the immunoreactivity of some antigens). Wash thoroughly the remaining PFA with PB.</li>
		<li>Cut the brain following indications of Figure 1d and glue the resulting block to the holder of a vibratome using cyanoacrylate.</li>
		<li>Collect 30 &#181;m-thick serial coronal sections using a vibratome. Store the brain slices in 24-multiwell plates with PB at 4 &#176;C. To prevent contamination, 0.05% sodium azide can be added to the PB solution.</li>
	</ol>
	</li>
	<li>Immunohistochemistry
	<ol>
		<li>Incubate the free-floating sections in blocking buffer (PB with 0.05% sodium azide, 1% glycine, 5% normal goat serum, and 0.1% Triton X-100) for 1h at RT with gentle shaking in a rocking platform.</li>
		<li>Carefully remove the blocking buffer with a pipette, add an appropriate dilution of anti-GFP rabbit primary antibody (see Table of Materials) in blocking buffer and incubate tissue with this dilution for 48 hr at 4 &#176;C with gentle shaking.</li>
		<li>Wash off the primary antibody solution a minimum of 3 times with PB, one wash every 10 min.</li>
		<li>Incubate the free-floating sections with a suitable dilution of fluorophore-conjugated secondary antibodies) in blocking solution (see Table of Materials) for 1 hr at RT and gentle shaking. Protect the sections from direct light during the incubation.</li>
		<li>Wash off the secondary antibody solution with PB, 3 times once every 10 min, and counterstain the tissue by incubating the sections with DAPI (4&#39;,6-diamidino-2-phenylindole) at 1 mg/ml in water for 5 min. Wash off the DAPI solution by rinsing twice and quickly with water.</li>
		<li>Gently place the sections on a microscope slide using a fine paint brush. Pour a few drops of mounting medium for fluorescent preparations (see Table of Materials) over the tissue and carefully place a coverslip on top, checking that the mounting solution is correctly distributed over the entire surface and there are no bubbles. Gently squeeze down the coverslip to drain the excess of mounting medium.</li>
		<li>When the mounting solution dries out (2-16 hr), analyze the sample by confocal laser scanning microscopy with the 488 nm laser.</li>
	</ol>
	</li>
</ol>

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<li>Bomsel, M., Alfsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Entry+of+viruses+through+the+epithelial+barrier%3A+pathogenic+trickery%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Entry of viruses through the epithelial barrier: pathogenic trickery.</a> Nat Rev Mol Cell Biol. 4 (1), 57-68 (2003).</li>
<li>Castellani, S., Di Gioia, S., Trotta, T., Maffione, A. B., Conese, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+lentiviral+vector-mediated+transduction+on+the+tightness+of+a+polarized+model+of+airway+epithelium+and+effect+of+cationic+polymer+polyethylenimine%5BTitle%5D)+AND+%22J+Biomed+Biotechnol%22%5BJournal%5D)">Impact of lentiviral vector-mediated transduction on the tightness of a polarized model of airway epithelium and effect of cationic polymer polyethylenimine.</a> J Biomed Biotechnol. , (2010).</li>
<li>Bonazzi, M., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impenetrable+barriers+or+entry+portals%3F+The+role+of+cell-cell+adhesion+during+infection%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Impenetrable barriers or entry portals? The role of cell-cell adhesion during infection.</a> J Cell Biol. 195 (3), 349-358 (2011).</li>
<li>Padmashali, R., You, H., Karnik, N., Lei, P., Andreadis, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adherens+junction+formation+inhibits+lentivirus+entry+and+gene+transfer%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Adherens junction formation inhibits lentivirus entry and gene transfer.</a> PLoS One. 8 (11), (2013).</li>
<li>Yamashita, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subventricular+zone-derived+neuroblasts+migrate+and+differentiate+into+mature+neurons+in+the+post-stroke+adult+striatum%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Subventricular zone-derived neuroblasts migrate and differentiate into mature neurons in the post-stroke adult striatum.</a> J Neurosci. 26 (24), 6627-6636 (2006).</li>
<li>Platt, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-Cas9+knockin+mice+for+genome+editing+and+cancer+modeling%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">CRISPR-Cas9 knockin mice for genome editing and cancer modeling.</a> Cell. 159 (2), 440-455 (2014).</li>
</ol>

All the manipulations were made in a biosafety level 2 room. Animal protocols were approved by the ethics committee of the University of Valencia and were all in compliance with European directive 2010/63/EU.

LVs offer important advantages over other viral systems for the genetic modification of adult NSCs16,18. Stereotaxic delivery of lentiviruses to the V-SVZ niche represents an efficient method to label and trace infrequently dividing B1-NSCs overcoming the limitations of other commonly used methods such as BrdU, which is diluted after multiple cell divisions, or retrovirus, which only target cells that are proliferating at the moment of application. LVs, together with adenoviruses, can infect cells independently of their cycling status and this is especially relevant for cells that do not cycle, such as ependymal cells, or are relatively quiescent, and thus divide rarely, like adult NSCs16,18. The transgenes become stably integrated and expressed with high efficiency allowing morphological characterization of transduced cells and their progeny.
NSCs are harbored in a highly specialized niche with neighboring cells such as ependymal or vascular cells as well as their own cell progeny thus creating a tightly regulated environment2. Like many fetal NSCs, B1 cells lie next to the ventricle and the ventricle contact its fluid through small, apical processes that end up in a single non-motile primary cilium immersed in the ventricular CSF1 (Figure 2a). This detailed organization can be exploited to specifically target ependymal cells or all cells in the niche through the control of the viral titer and delivery area. Adherens and tight junctions in polarized epithelia have been shown to hinder entry of foreign viruses into cells and tissues, sometimes due to the restricted location of viral entry receptors (i.e. CAR receptors) in the junctional complexes. Disruption of intercellular adhesion therefore favors infection by different types of viruses, including the lentivirus (LV). Interestingly, LVs have been shown to disrupt junctional complexes when used at high concentrations in lung epithelium20-23. This could explain previous reports of infections of B1-NSCs by LVs injected into the lateral ventricle24.
Since B1-NSCs produce progeny that migrate anteriorly and dorsally, GFP-positive cells can be found at all levels of the RMS as well as in the OB and corpus callosum where they terminally differentiate into neurons and oligodendrocytes, respectively. However, we do not use the reporter expression to determine effects of the genetic modification on the rates of neurogenesis or oligodendrogenesis. On one hand, the corpus callosum is in the path of the needle track and, therefore, some cells that appear labeled may be the result of a direct infection during the injection process. On the other hand, because all types of cells in the V-SVZ can be infected it is not possible to distinguish whether the labeled OB neurons are the result of a genetic modification in the B1 cells or their progenitor derivatives. In the case of experiments in which only the ependyma is infected, B1 cells and their progeny are not labeled with the reporter and therefore neurogenesis or oligodendrogenesis cannot be traced. However, one can take advantage of BrdU retention to analyze B1-NSC dependent neurogenesis to the OB or oligodendrogenesis to the corpus callosum as well as the proliferative activity of slowly-dividing NSCs in the V-SVZ of the infected mice. For example, the mice can be injected with BrdU following previously published protocols17 10 days after the infection with the LVs and allowed to survive for 21 days. BrdU detection in the OB/corpus callosum can then be used to determine the rate of neurogenesis/oligodendrogenesis, whereas BrdU retention in the V-SVZ can be taken as an estimate of the numbers of activated B1-cells13.
The procedure described here can be used for the expression of genes of interest for gain-of-function experiments but can also be used for the delivery of silencing constructs based on RNA interference or for the delivery of cre recombinase constructs to inactivate germ-line transmitted floxed alleles in the V-SVZ cells. In addition, this technique could also be employed for CRISPR-mediated genome editing if injection of lentivirus-single guide RNAs is used in combination with a Cre-dependent Rosa26 Cas9 knockin mouse25.
Critical step 1: lentivirus production and manipulation. In vivo gene transfer applications with LVs into specific brain areas require high titer vector preparations, since only very small volumes can be injected stereotaxically without damaging the brain tissue. Lentiviral preparations need to be purified and efficiently concentrated before injection into the brain due to the fact that LVs are generated in transient co-transfection systems. The procedure described herein is biosafety level 2, therefore all procedures that require the manipulation of viruses are performed in BSL2 facilities. Research personnel need to be appropriately qualified and trained in all procedures. In addition, surgery must be performed by fully trained investigators following routines approved by the local authorities for animal welfare, and should be done under aseptic conditions with properly sterilized instruments in biosafety level 2 areas. Adequate pre-operative and post-operative care of animals must be carried out and should be in accordance with established veterinary medical and nursing practices. More specifically, all precautions must be taken to minimize the pain and stress inflicted to the animal including administration of anesthesia and analgesia. Every step concerning the use of viruses in animals must be authorized by the institutional authorities and performed in accordance with local regulations. Personal protective equipment must also be worn, including a gown, double gloves and suitable eye protection. Finally, all tools and surfaces that could have been in contact with viruses must be thoroughly decontaminated according to the facility&#180;s approved disinfection practices (by wiping with 70% ethanol, 10% bleach and/or autoclaving).
In our experience, the calcium phosphate transfection method is a very efficient and cheap technique of introducing DNA into 293T cells. However commercial DNA transfection reagents can also be used as per manufacturer&#39;s protocol in this step. This protocol is optimized for the collection of the viral supernatant at 30 hours after media replacement of the 293T cells. Lentiviral supernatant can also be collected 24 and 48 hr after media replacement. However, in our experience a single collection at 30 hr produces a high-titer lentivirus vector.
Critical step 2: practical example of the calculations of the vector titer using the formula: % GFP+/100 x number of cells infected x dilution factor (DF) = transducing units (TU)/ml. In this example, 5 x 104 cells (293T) are seeded the previous day allowing for the transduction of 105 cells. After obtaining the % of transduced cells using flow cytometry as described in the protocol, two values of transduced cells are chosen for the calculations, those of which correspond to the dilutions at which transduction is neither saturated nor too low. For example: Dilution Factor 10-6 yield 8.08% cells infected, and dilution factor 10-5 yield a 61.78% cells infected. The titer is the average of the values rendered from applying the formula using the values of the cells transduced and its corresponding dilution factor. In this example:
Titer for the dilution factor 10-6 = (8.08/100) x 100,000 x 106 = 8.08&#183;109 TU/ml
Titer for the dilution factor 10-5 = (61.78/100) x 100,000 x 105 = 6.178&#183;109 TU/ml
Average = [(8.08 + 6.178)/2] &#183; 109 = 7.1 &#183; 109 TU/ml
Critical step 3: stereotaxic injection. The method described here relies on the use of low volumes of high titers of LVs stereotaxically guided delivery into specific locations. The stereotaxic coordinates given in this protocol have been established for C57/BL6 mice and may not be suitable for other mouse strains. Stereotaxic coordinates should be determined for each strain and age using a dye like fast green prior to the experiment. To do this, the dye is injected and after the mouse is sacrificed, the brain is extracted and frozen for 1 hrat -20 &#176;C. Once the brain is hardened, it can be sliced with a blade at the site of the injection and observed under a dissecting microscope to determine whether the injection site is optimized for specific experimental purposes.
Critical step 4: post-survival times. Post-survival times will be different depending on the site of injection. A long-term survival time allows brain tissue to recover from the injection lesion and, more importantly, cells observed in the RMS and OB after 60 days are derivatives of the relative quiescent B1-NSCs. Injections into the lateral ventricle do not physically harm the V-SVZ. Therefore, a 15 day period is enough to ensure expression of the transgenes by ependymal cells.
Critical step 5: post-fixation times. Excessive post-fixation of the brain tissue may generate masking of some antigens, due to the crosslinking activity of the reagents, such as paraformaldehyde. These are good at preserving cell structure, but may reduce the antigenicity of some proteins as the crosslinking can obstruct antibody binding to the epitopes. Antigen retrieval techniques may be required, particularly if there is a long fixation incubation time or if a high percentage of crosslinking fixative is used. Alternatively, shorter periods of post-fixation of 1 hr have proved sufficient in our experience to maintain the structure of the tissue as well as ensuring a good antigenicity within the cells without the use of antigen retrieving procedures.

The murine ventricular-subventricular zone (V-SVZ), in the walls of the lateral ventricle facing the striatum, is a very active germinal region in which a continual process of progenitor cell replication and differentiation results in the persistent production of olfactory bulb (OB) interneurons and corpus callosum oligodendrocytes1. The lifelong generation of these cells appears to be supported by the presence in this region of neural stem cells (NSCs; also called B1 cells), which express the astrocytic antigen glial fibrillary acidic protein (GFAP) and stem cell markers such as nestin, Id1 and Sox22. GFAP-expressing B1 cells generate transit amplifying progenitor (TAP) cells (C cells), which express transcription factors Dlx2 (distal-less homeobox 2) and Ascl1 (mammalian achaete-schute homolog 1) and divide rapidly a few times before they give rise to migrating neuroblasts (A cells) or oligodendroblasts3. Newly-generated proliferative neuroblasts migrate anteriorly, forming the rostral migratory stream (RMS) to the OB, where they integrate into the granular and glomerular layers as differentiated inhibitory interneurons. Migrating young oligodendroblasts move to the CC, where they become immature NG2-positive cells that continue to divide locally or differentiate into mature myelinating oligodendrocytes1,4.
B1 cells, which derive from fetal radial glial cells, retain the elongated and polarized morphology of their predecessors and exhibit a highly specialized relationship with their niche. They span between the ependyma which lines up the ventricle and the network of blood vessels that irrigate the V-SVZ niche. The small apical process of B1 cells intercalates among multiciliated ependymocytes and ends in a single non-motile primary cilium, whereas their basal process extends long distances to approach the planar vascular plexus that irrigates this niche ending in the basal lamina of the plexus capillaries2,5-8 .
The most reliable way to distinguish B1-NSCs from non-neurogenic astrocytes, which are also GFAP+, in the intact V-SVZ niche is based on whole-mount preparations of the ventricle lateral wall and their analysis by 3-D confocal microscopy after immunostaining for GFAP to label the thin B1-NSC apical process, &#946;-catenin to delineate cell membranes, and either &#947;-tubulin as a marker of cilial basal bodies or acetylated &#945;-tubulin to label the extent of each cilium5,8. Observations of these whole-mounts from the ventricular surface have indicated that B1 and ependymal cells are arranged in &#34;pinwheels&#34;5, in which the uniciliated apical processes of one or several GFAP+ B1 cells are encircled by a rosette of multiciliated ependymal cells.
The characteristic morphology of B1 cells correlates with experimental evidence indicating that blood vessels/endothelial cells and ventricular cerebrospinal fluid (CSF) constitute regulated sources of soluble signals acting on NSCs2,6,9-11. At the ventricular surface, homotypic and heterotypic apico-lateral interactions involving ependymal and B1 cells include tight junctions and adherens junctions5,12. Moreover, adhesion molecules implicated in the junctional complexes between B1 and ependymal cells, such as N-cadherin and V-CAM, have been shown to regulate not only the highly organized positioning of B1 in the V-SVZ niche, but also their quiescence12,13. The ependymal-B1 cell monolayer appears to act as a diffusion barrier allowing the regulated flux of water and small molecules from the CSF, but restricting the intercellular passage of large proteins10,11. Experimental evidence indicates that the uniquely positioned B1 cell apical cilium could play a role as a sensor of signaling polypeptides present in the CSF2,5-7. Ependymal cells are, per se, also a source of soluble and membrane-bound signals with a role in the regulation of NSC behavior14,15.
Traceable nucleosides, such as bromo-deoxyuridine (BrdU), or retroviruses have been widely used to label progenitor cells, including NSCs, in vivo. However, these methods are not optimal for long-term fate tracing because BrdU signals dilute through repeated cell divisions and retroviruses appear to preferentially target transiently amplifying cells due to their requirement of cell proliferation for transduction16,17. To examine NSC physiology in vivo, including interactions with niche components, it is crucial to establish a method to label and trace rarely dividing cells, as B1-NSCs are largely quiescent and their neighboring ependymal cells never divide under physiological conditions3. Here, we show that lentiviral vectors (LVs) allow for high-efficiency gene marking and long-term modification of adult NSCs and non-dividing ependymal cells, due most reasonably to their ability to transduce and to integrate into the genome of target cells in a cell cycle-independent way. Moreover, we show how the route of delivery and viral titer help to specifically transduce ependymal cells, but not B1 cells thereby allowing the analysis of niche-dependent, ependymal effects on NSCs.

<table><tbody><tr><td>&lt;em&gt;&lt;strong&gt;Part 1: Generation of LV for in vivo delivery.&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Equipment:&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima XL-100K</td><td></td></tr><tr><td>Ultracentrifuge rotor</td><td>Beckman Coulter</td><td>SW-28</td><td></td></tr><tr><td>Ultracentrifuge rotor</td><td>Beckman Coulter</td><td>SW-55</td><td></td></tr><tr><td>Ultracentrifuge tubes&amp;nbsp;</td><td>Beckman Coulter</td><td>358126</td><td>25 mm x 89 mm</td></tr><tr><td>Ultracentrifuge tubes&amp;nbsp;</td><td>Beckman Coulter</td><td>326819</td><td>13 mm x 51 mm</td></tr><tr><td>Ultracentrifuge adapters</td><td>Beckman Coulter</td><td>358156</td><td></td></tr><tr><td>6-well plate</td><td>SPL</td><td>PLC-30006</td><td></td></tr><tr><td>24-well plate</td><td>SPL</td><td>PLC-30024</td><td></td></tr><tr><td>10 cm dish</td><td>SPL</td><td>PLC-20101</td><td>100 x 20 style</td></tr><tr><td>FACS tubes</td><td>Afora</td><td>DE400800</td><td>12 mm x 75 mm, 5 ml</td></tr><tr><td>Cup sterile FACS filter</td><td>BD</td><td>340626</td><td>30 &amp;micro;m</td></tr><tr><td>Nitrocellulose filter</td><td>Millipore</td><td>SCGPU05RE</td><td>0.22 &amp;mu;m&amp;nbsp;</td></tr><tr><td>Flow cytometer</td><td>BD</td><td>LSR Fortessa</td><td>Blue laser 488 nm</td></tr><tr><td>Steritop filter</td><td>Biofil</td><td>FPE-204-500&amp;nbsp;</td><td>0.22 &amp;micro;m</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Reagents:&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>pMDLg/pRRE plasmid&amp;nbsp;</td><td>Addgene</td><td>#12251</td><td>Core packaging plasmid</td></tr><tr><td>pRSV.REV plasmid&amp;nbsp;</td><td>Addgene</td><td>#12253</td><td>Core packaging plasmid</td></tr><tr><td>pMD&lt;sub&gt;2&lt;/sub&gt;G plasmid&amp;nbsp;</td><td>Addgene</td><td>#12259</td><td>Envelope plasmid</td></tr><tr><td>pRRL-SIN-PPT.PGK.EGFP.Wpre plasmid&amp;nbsp;</td><td>Addgene</td><td>#12252</td><td>Transfer vector plasmid</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium&amp;nbsp;</td><td>Biowest</td><td>L0101-500</td><td>For HeLa cell culture</td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium&amp;nbsp;</td><td>Life technologies</td><td>12440-053</td><td>For 293T cell culture</td></tr><tr><td>Tris-EDTA (TE)</td><td></td><td></td><td>Tris-HCl (sigma, T5941), 0.1 mM EDTA (sigma, E5134), pH 7.6,&amp;nbsp; DNAse/RNAse-free, 0.2 &amp;micro;m sterile-filtered</td></tr><tr><td>2x HBS</td><td></td><td></td><td>0.28 M NaCl (Sigma, S7653), 0.05 M HEPES (Sigma, H7523), 1.5 mM anhydrous Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4 &lt;/sub&gt;(Sigma, S7907) in dH&lt;sub&gt;2&lt;/sub&gt;O (preferably not MilliQ). Adjust pH to 7.0 with NaOH solution (Calbiochem, 567530).</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Biowest</td><td>S181B-500</td><td>Stock solution at 100x, used to prepare HeLa and 293T culture medium at a final concentration of 10x.</td></tr><tr><td>Glutamine</td><td>Sigma-Aldrich</td><td>G7513-100</td><td>Stock solution at 200 mM, used to prepare HeLa and 293T culture medium at a final concentration of 6 mM.</td></tr><tr><td>Sodium pyruvate</td><td>Life technologies</td><td>11360-039</td><td>Stock solution at 100 mM, used to prepare HeLa and 293T culture medium at a final concentration of 1 mM.</td></tr><tr><td>GlutaMAX Supplement</td><td>Life technologies</td><td>35050-061</td><td>Used to prepare 293T culture medium at a final concentration of 1%.</td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma-Aldrich</td><td>P4458</td><td>Stock solution contains 5,000 units/ml penicillin and 5 mg/ml streptomycin. Used to prepare HeLa and 293T culture medium at a final concentration of 1%.</td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies</td><td>25200-056</td><td>With phenol red, contains 2.5 g porcine trypsin and 0.2 g EDTA 4Na/L HBSS.</td></tr><tr><td>Phosphate buffered saline&amp;nbsp; (PBS)</td><td>Sigma-Aldrich</td><td>D1408</td><td>Without calcium chloride and magnesium chloride, 10x, liquid, sterile-filtered, suitable for cell culture. Stock solution used to prepare 1x PBS in cell culture grade water.</td></tr><tr><td>Polybrene (hexadimethrine bromide)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H9268</td><td>Powder. Prepare a 1,000x stock solution at 8 mg/ml in dH&lt;sub2&lt;/sub&gt;O</td></tr><tr><td>Paraformaldehyde EM grade 16%</td><td>EM Sciences</td><td>15710</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Part 2: Sterotaxic injection of LV into the SEZ proper or the lateral ventricle.&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Equipment:&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Vernier stereotaxic instrument</td><td>NeuroLab, Leica</td><td>39463001</td><td>http://www.leicabiosystems.com/</td></tr><tr><td>Cunningham mouse and neonatal rat adaptor</td><td>NeuroLab, Leica</td><td>39462950</td><td></td></tr><tr><td>Syringe holder</td><td>KD Scientific</td><td>KDS-311-CE</td><td></td></tr><tr><td>33 gauge syringe</td><td>Hamilton</td><td>P/N&amp;nbsp;&amp;nbsp;&amp;nbsp; 84851/00</td><td>#85RN</td></tr><tr><td>Electric drill</td><td>Fine Science Tool</td><td>98096</td><td></td></tr><tr><td>Thermal blanket</td><td>Ufesa</td><td>AL5512/01</td><td>230-240 V, 100-110 W, type C_AL01</td></tr><tr><td>Shaver&amp;nbsp;</td><td>Jata</td><td>MP373N</td><td>Model: beauty, 3 V, 300 mA, type HT-03.</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Reagents:&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Medetomidine</td><td>Esteve</td><td>DOMTOR&amp;nbsp;</td><td>Comercial solution at 1 mg/ml.&amp;nbsp;</td></tr><tr><td>Ketamine</td><td>Merial</td><td>Imalgene 500&amp;nbsp;</td><td>Comercial solution at 50 mg/ml</td></tr><tr><td>Medetomidina/ketamine mixture</td><td></td><td></td><td>Prepare a working mixture of medetomidine at a final concentration of 0.2 mg/ml dilution and ketamine at a final concentration of 15 mg/ml in saline solution. Use as anesthesia injecting a volume to get a final concentration of 0.5-1 mg medetomidina per kg body weight and 50-75 mg ketamine per kg body weight</td></tr><tr><td>Butorphanol</td><td>Pfizer</td><td>Torbugesic</td><td>Stock solution at 10 mg/ml. Used as analgesia at 1 mg/ml in saline solution.</td></tr><tr><td>Atipamezole</td><td>Esteve</td><td>Antisedan</td><td>Stock solution at 5 mg/ml, used in a final concentration of 0.5 mg/ml in saline solution to exit from anesthesia.</td></tr><tr><td>0.9% saline solution</td><td>Braun</td><td>13465412</td><td></td></tr><tr><td>Histoacryl</td><td>Braun</td><td>1050052</td><td>Topical skin adhesive</td></tr><tr><td>HydroGel</td><td>Clear H&lt;sub&gt;2&lt;/sub&gt;O</td><td>70-01-5022</td><td></td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark</td><td>34120</td><td>11 cm x 21 cm</td></tr><tr><td>Bleach/Virkon</td><td>Dupont</td><td></td><td></td></tr><tr><td>Surgical marker pen</td><td>Staedler</td><td>313-9</td><td>Permanent lumocolor</td></tr><tr><td>Ophthalmic lubricant &amp;nbsp;</td><td></td><td>SICCAFLUID</td><td>0.5 g/dosis, carbomer 974P</td></tr><tr><td>Povidone-iodine</td><td>Betadine</td><td>694109.6</td><td>10% povidone-iodine</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Part 3: Histological analysis.&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Equipment:&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Automatic peristaltic pump</td><td>Cole-Parmer Inst. Co.</td><td>HV-07524-55</td><td>Masterflex L/S variable-speed economy drive, 1.6-100 rpm, 230 V</td></tr><tr><td>Pump head</td><td>Cole-Parmer Inst. Co.</td><td>HV-07518-00</td><td>Masterflex L/S Easy-Load pump head for precision tubing; PSF housing, CRS rotor</td></tr><tr><td>Silicone tube</td><td>Cole-Parmer Inst. Co.</td><td>HV-96410-16</td><td>Platinum L/S 16</td></tr><tr><td>Scalp vein set</td><td>Vygon V-green</td><td>70246.05T</td><td>25 G, 30 cm tube length</td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FluoView FV10i</td><td></td></tr><tr><td>Hot plate</td><td>Tehtnica</td><td>SHP-10</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Reagents:&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phosphate buffer (PB)</td><td></td><td></td><td>0.2 M PB: 0.2 M Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (Sigma, S7907) and 0.2 M NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;(Panreac, 141965.1211) in dH&lt;sub&gt;2&lt;/sub&gt;O, adjust pH to 7.2-7.4</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Panreac</td><td>141451.1211</td><td>Prepare fresh every time. Heat dH&lt;sub&gt;2&lt;/sub&gt;O up to 55&amp;ndash;60 &amp;deg;C using a hot plate placed in a fume hood and pour PFA powder while stirring to obtain an 8% solution. The solution is cloudy white as PFA does not dissolve easily. Add 1N NaOH drop by drop just until the solution clears. Cool down, filter through Whatman paper and add an equivalent volume of 0.2 M PB.</td></tr><tr><td>Saline solution</td><td></td><td></td><td>0.9% NaCl in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Superglue</td><td>LOCTITE</td><td>767547</td><td></td></tr><tr><td>Sodium azide</td><td>Panreac</td><td>122712.1608</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G7126-100</td><td></td></tr><tr><td>Normal goat serum</td><td>Millipore</td><td>S30-100</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td>Detergent</td></tr><tr><td>Anti-GFP rabbit antibody</td><td>ROCKLAND</td><td>600-401-215</td><td>Use at a 1:500 dilution</td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L) Antibody</td><td>Molecular probes</td><td>A-21206</td><td>Use at a 1:750 dilution</td></tr><tr><td>6-Diamindino-2-phenylindole dihydrochloride hydrate (DAPI)</td><td>Sigma-Aldrich</td><td>D9542</td><td>Fluorescent nuclear staining. Use at 2 mg/ml in ddH&lt;sub&gt;2&lt;/sub&gt;O. Keep in the dark at 4 &amp;deg;C.</td></tr><tr><td>Fluoromount-G</td><td>EM Sciences</td><td>17984-25</td><td>Mounting medium for fluorescent preparations</td></tr></tbody></table>

stable and efficient genetic modification of cells in the adult mouse v-svz for the analysis of neural stem cell autonomous and non-autonomous effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

LV-mediated gene delivery system can be used for the long-term in vivo transduction of cells in the adult mouse V-SVZ, allowing their tracking and genetic modification during proliferation, migration and differentiation. The infection and the expression are highly effective and yield numerous cells that can be easily distinguished among other non-infected cells by the expression of the reporter included. We have thus far visualized transduced cells with GFP fluorescent reporters, driven by the ubiquitously expressed phosphoglycerate kinase promoter, but other reporters or protein tags can also be used. We routinely use antibodies to GFP instead of relying on the direct detection of the reporter emission as it yields a stronger fluorescent signal. Moreover, the brains can be dissected fresh and immersed in fixative but much better results are obtained by transcardial perfusion.
Purified, concentrated vector stocks are slowly injected under stereotaxic guidance into the V-SVZ at its limit with the striatum (Figures 2a, b). Two months after injection, numerous GFP+ cells were found in the V-SVZ and in an approximate 1 mm radius from the injection site (Figures 2a, b). Infection with LVs results in the transduction of all types of cells in the V-SVZ and expression of the reporter by ependymal cells indicates that LVs can also efficiently target non-dividing cells (Figure 2c; see also 15). GFP-labeling pattern in the V-SVZ was similar to that found after 2 weeks18. No sign of tissue reaction, however, was detectable at 2 months post injection since the long period allows the recovery of the tissue from the injury inflicted by the surgery. During the two-month period, infected TAP cells progress into A-neuroblasts that migrate out the zone towards the OB. Because this process takes only a few days3, the presence of GFP+ neuroblasts in the RMS 60 days after the infection indicates that LVs also target neurogenic B1-NSCs (Figure 2d). The transduction of B1, C, and A cells at the time of injection yields GFP+ interneurons in the OB (Figure 2e).
Injection of a small volume of high-titer concentrated LV stocks into the lateral ventricle results in the infection of ependymocytes only (Figures 2f-h). Most ependymal cells, which contain the calcium binding protein S100&#946;, express the reporter after only 15 days from infection (Figure 2i). In contrast to the injection into the V-SVZ/striatal limit, no GFP+ cells were observed subependymally located or in the RMS or OB using this procedure (Figures 2h, j). Both types of infections can be analyzed in sections through the V-SVZ as explained. They can also be analyzed in whole-mount preparations of the lateral ventricle wall (see protocol in 8).
Recently, we have described how N-cadherin mediated cell-cell adhesion of B1 cells to ependymocytes regulates their quiescence. We have used the procedure described here to deliver LVs carrying cadherin dominant-negative constructs that resulted in inactivation of N-cadherin. We have demonstrated that loss of N-cadherin in the NSCs and ependymal cells or in ependymal cells only promoted an increase in the number and cell cycle entry of NSCs as a result of the disruption of homotypic cell-cell interactions (Figures 3a, b; see 15).
<img alt="Figure 2" src="/files/ftp_upload/53282/53282fig2.jpg" /> 
Figure 2: Reporter detection by immunofluorescence in sections of injected mice. (a) Schematic representation of the adult sub-ventricular zone niche. A simplification of the cellular components of the niche is depicted. B1 cells are polarized and exhibit a specialized relationship with the niche. They span between the ependyma which lines up the ventricle and the network of blood vessels that irrigate the V-SVZ niche. The small apical process of B1 cells intercalates among multiciliated ependymocytes and ends in a single non-motile primary cilium, whereas their basal process extends to approach the planar vascular plexus that irrigates this niche ending in the basal lamina of the plexus capillaries. B1 cell derivatives are intermediate progenitors that actively proliferate in the surroundings of blood vessels and migrating chains of neuroblasts towards the olfactory bulbs in contact with niche astrocytes. (b) Schematic of a coronal section through one hemisphere showing the level of injection and the position of the syringe needle for injections at the V-SVZ/striatum border. (c) Micrograph taken at the same level shown in the schematic stained with DAPI (gray; left) and for the immunodetection of GFP (green; right) 60 days after the injection. The dotted lines indicate the limits of the corpus callosum. (d) Higher magnification of an immunofluorescent detection of the GFP reporter injected in the V-SVZ vicinity or striatum border in a coronal section through the V-SVZ. The dotted lines indicate the limits of the V-SVZ. Note that many cells are infected in the V-SVZ niche. Ependymal cells can be recognized as cuboidal cells limiting the ventricle lumen. (e) Immunofluorescence for GFP (green) and the neuroblast marker PSZ-NCAM (red). Note the presence of doubly positive cells, indicating the generation of neuroblasts from NSCs that were infected 60 days before. White arrowheads point at neuroblasts. Insets correspond to the region enclosed within the white square. (f) Schematic of a coronal section through one hemisphere showing the level of injection and the position of the syringe needle for injections at the lateral ventricle. (g) Micrograph taken at the same level shown in the schematic stained with DAPI (in grey; left) and for the immunodetection of GFP (in green; right) 15 days after the injection. The dotted lines indicate the limits of the corpus callosum. (h) Higher magnification of an immunofluorescent detection of the GFP reporter injected in the ventricle in a coronal section through the V-SVZ. The dotted lines indicate the limits of the V-SVZ. Note that only ependymal cells are infected. (i-j) Immunofluorescence for GFP and the ependymal cell marker S100&#946;. Note the presence of numerous doubly positive cells along the dorsal (i) and ventral (j) V-SVZ. Arrowhead points at an astrocyte in the striatal parenchyma, also labeled with S100&#946;. k. Coronal section through the OB stained with DAPI (gray; left) and immunodetection of GFP (green; right) in the region enclosed by the white square 60 days after a V-SVZ/striatum injection. Notice numerous labeled neurons. (l) Coronal section through the OB stained with DAPI (gray; left) and immunodetection of GFP (in green; right) region enclosed by the white square 45 days after a lateral ventricle injection. Notice that there are no OB neurons labeled. cx, cerebral cortex; lv, lateral ventricle; str, striatum; GL, glomerular layer; GCL, granule cell layer; MCL, mitral cell layer. Scale bars: c, g = 500 &#181;m; d, e, h-j = 10 &#181;m; k-l = 250 &#181;m; 100 &#181;m.
<img alt="Figure 3" src="/files/ftp_upload/53282/53282fig3.jpg" /> 
Figure 3: Adult NSCs proliferate more actively when they become detached from their niche. (a) Confocal micrographs of sections through the V-SVZ of mice that received striatal injections of empty LVs (upper panel) or LVs carrying a dominant-negative cadherin construct (bottom panel), immunostained for the detection of the LV reporter GFP (green), the NSC marker GFAP (red), the cell cycle marker Ki67 (cyan), and DAPI (blue) 60 days after the infections. Independent panels for each marker are shown (grey; right). (b) Confocal micrographs of sections through the V-SVZ of mice that received injections of empty LVs (upper panel) or LVs carrying a dominant-negative cadherin construct (bottom panel) into the lateral ventricle immunostained for the detection of the LV reporter GFP (green), the NSC marker GFAP (red), the cell cycle marker Ki67 (cyan), and DAPI (blue), 15 days after the infections. Independent panels for each marker are shown (gray; right). White arrows point at GFAP/GFP double positive cells and white arrowheads point at GFAP/Ki67/GFP triple positive cells whose nuclei in both cases are indicated by a yellow asterisk. Quantitative analysis indicated more cycling NSCs when the N-cadherin levels were decreased in the NSCs themselves or only in ependymal cells15. Scale: 10 &#956;m.

Watch this Scientific Journal Video about Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects at JoVE

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

stable-efficient-genetic-modification-cells-adult-mouse-v-svz-for

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.2K Views.  Spanish National Cancer Research Centre (CNIO). Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Video: Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

Neuroscience

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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

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.

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

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Rat "Brain Milking" for Neural Stem and Oligodendrocyte Progenitor Cell Isolation

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

Author Spotlight: Collecting Neural Stem and Progenitor Cells from Live Animals Using a Novel Brain Milking Protocol 

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

Author Spotlight: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

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