This work was supported by the Center for Regenerative Therapies, the Medical Faculty of the TU Dresden, and the DFG Collaborative Research Center SFB655 (subproject A20). BA was supported by a fellowship of the DIGS-BB program, Dresden. We thank Drs. Noriko Osumi, Tadashi Nomura, Dieter Chichung Lie, and Ravi Jagasia for precious advice during the setting up of in utero electroporation and stereotaxic viral injection.

Preliminary note
Surgery must be performed by fully trained investigators upon approval from the local authorities for animal welfare. All precautions must be taken to minimize the pain and stress inflicted to the animal including deep anesthesia, administration of postoperative analgesia and, for operations lasting more than 10 min, application of an eye lubricant to prevent corneal dehydration and blindness. Anesthetized mice must be kept constantly warm at 37 &deg;C until full recovery and every surgical tool and solution must be sterile. Although the use of a biological hood is not strictly necessary, the operator must take all reasonable precautions to minimize the possibility of infecting the animal during the operation by wearing a lab coat, mask, and gloves. Similarly, every step concerning the generation and use of viruses must be authorized and must be performed in S2 areas according to the regulations of the local authorities. Finally, a thorough decontamination of all tools and surfaces that could have been in contact with viruses must be performed according to approved facility disinfection practices (for HIV by wiping with 70% Et-OH and/or autoclaving).
Part 1: Expansion of Embryonic NSC
1. In utero electroporation
<ol>
 <li>After cloning the transgenes (i.e. cdk4/cyclinD1) in pCMS-EGFP (Clontech) or pDSV-mRFPnls vectors 16, purify the plasmids using EndoFree kit and resuspend in sterile PBS to a concentration of 3-5 &mu;g/&mu;l. Soon before surgery, mix the plasmids with 0.05% FastGreen in PBS at a final ratio of ca. 4:4:1 and centrifuge the mixture for 2 min at 16,000 x g to remove all precipitates.</li>
 <li>Load the DNA mixture into a previously pulled borosilicate glass capillary. Pulling parameters using a P-97 pipette puller are: pull: 200; vel: 140; time: 140. Heat is given by a ramp test and depends on the specific lot of capillaries being used. Mount the capillary on the nozzle of the PicoPump that is near to the in utero electroporation platform (Figure 1A) and, under a stereomicroscope, bend its tip and nick it at the inflection point.</li>
 <li>Sterilize all surgery tools in a dry glass bead sterilizer and deeply anesthetize a pregnant mouse at day 12-15 of gestation using an isoflurane vaporizer. Place the animal in supine position on a heating platform set at 37 &deg;C and keep under constant isoflurane administration through a nose cone.</li>
 <li>Shave the skin of the abdomen, disinfect with Betaisodona multiple times, eventually wiping in between with 70% ethanol, and inject buprenorphine (diluted in PBS) subcutaneously at 0.1 mg/kg concentration as pre-emptive analgesic. Using fine scissors, make a 2 cm longitudinal cut of the skin and, subsequently, of the underlying muscular wall to access the peritoneal cavity. Dispense in the peritoneal cavity ca. 2 ml of IUE solution (D-PBS containing 100 U/ml of pen/strep) prewarmed at 37 &deg;C and keep the solution on a heating block.</li>
 <li>Cover the mouse with sterile drap containing a fissure from which the uteri will be removed. Retract the incision using a tungsten retractor, identify the uterus and pull it out holding it with forceps between adjacent embryos (Figure 1B; left) and finally lay it down on the sterile drap. During the whole operation, rinse the uterus with IUE solution to moisturize the organ and body cavity and prevent dehydration of the animal. </li>
 <li>Handle the uterus carefully holding it between thumb and index and turn one embryo until its head is visible and oriented towards the operator. Identify the telencephalic hemispheres and inject one of them from the dorso-lateral side. Release 1-2 &mu;l of the DNA solution using the footswitch of a PicoPump until the ventricle is outlined by FastGreen (Figure 1B; right). </li>
 <li>Place the anode of the electrodes on the injection site and the cathode on the contralateral side (Figure 1C) and deliver 6 pulses of 30 V for 50 ms with 950 ms interval between each pulse using an electroporator generating square-shaped electric fields operated through a footswitch. Avoid placing the electrodes close to the placenta as this may cause hemorrhage and damage the embryo.</li>
 <li>When all embryos are injected and electroporated, place the uterus back into the abdominal cavity and close the muscular walls with a suture string suturing every 3-4 mm. The knots should be snug to the muscle but not too tight to allow a little swelling of the tissue. Finally, close the overlying skin using metal clips. Disinfect the skin with Betaisodona, remove the mouse from the isoflurane mask and transfer it in a housing cage when fully awake. Monitor the recovery of the mouse until locomotor behaviour is normal. </li>
</ol>
2. Processing of the sample
<ol>
 <li>One or more days after electroporation, sacrifice the mouse, collect the embryos and identify those correctly electroporated using a fluorescent stereomicroscope (Figure 1D). Dissect the brains on ice-cold PBS, and fix them overnight in 4% PFA (paraformaldehyde in phosphate buffer pH 7.4). Eventually, BrdU can be administered before sacrifice according to different paradigms to investigate cell cycle kinetics and cell proliferation 3.</li>
 <li>The brains are then processed for sectioning and immunostaining for the several markers of radial glia, basal progenitors and neurons (Pax6, Nestin, Tbr2, Tbr1, Tuj1 etc.) to evaluate the effect of the functional transgenes on targeted NSC and their progeny that are identified by the expression of the co-electroporated reporter gene. </li>
</ol>
<img src="/files/ftp_upload/4093/4093fig1.jpg" alt="Figure 1" /> 
Figure 1. In utero electroporation. (A) Schematic representation of an in utero electroporation platform. (B) Drawings showing the positioning of the pregnant mouse during surgery (right) and injection of the DNA/FastGreen mixture (blue) in the telencephalic hemisphere of a mouse embryo (left). (C) Scheme showing the placement of the electrodes in contact with the uterine walls with the anode (+) adjacent to the site of injection (blue). Arrows indicate the migration of the DNA towards the anode. (D) Picture of a mouse embryo 24 hr after electroporation with GFP plasmids at embryonic day 13. Dashed line demarcates the telencephalic hemisphere. Scheme in A modified from Calegari et al., 2004 17.
Part 2: Expansion of Adult NSC
1. Production of HIV-derived viral particles
<ol>
 <li>Day 1: plate 5 x 106 293T cells on a 10 cm dish with 8 ml of D-MEM containing 10% of heat inactivated fetal bovine serum and 100 U/ml of pen/strep (culture medium). Use 15 dishes for one viral preparation and culture overnight at 37 &deg;C, 5% CO2.</li>
 <li>Day 2: for every dish, dilute in 1 ml of plain D-MEM 5 &mu;g of each of 3 plasmids encoding for i) VSVG (vesicular stomatitis virus type G) envelope protein, ii) gag/pol (group specific antigen/polymerase), and iii) a polycistronic construct expressing the functional and reporter transgenes (i.e. cdk4/cyclinD1/GFP) previously cloned in a HIV-based transfer vector (p6NST90)9. Mix this solution with 1 ml of D-MEM containing 45 &mu;l of polyethylenimine that has been dissolved beforehand in PBS at 1 mg/ml stock solution, and incubate for 15-30 min at room temperature. In the meanwhile, remove the media from the cells and add 4 ml per dish of fresh D-MEM containing 15% of heat inactivated fetal bovine serum and 100 U/ml of pen/strep. Add the 2 ml of transfection mixture and culture overnight.</li>
 <li>Day 3: add 120 &mu;l of 500 mM Na-butyrate per dish to enhance the expression of the transgenes, mix gently and culture for 6-8 hr after which replace the transfection medium with 5 ml of culture medium.</li>
 <li>Day 4: collect the supernatants (ca. 70 ml total), pass through a 0.22 &mu;m filter and transfer in 2 polyallomer centrifuge tubes (35 x 89 mm). Ultracentrifuge at 110,000 x g for 90 min at 4 &deg;C using a swing rotor. Discard the supernatant, add 6 ml of PBS to each tube and incubate on ice for 1 hr. Resuspend the pellet and transfer it in one polyallomer centrifuge tube (14 x 95 mm). Ultracentrifuge at 110,000 x g for 90 min at 4 &deg;C using a swing rotor. Discard the supernatant, resuspend the pellet in 50 &mu;l of PBS, make 5 &mu;l aliquots and store at -80 &deg;C. Viral titer will be in the range of 108-109 cfu/ml. (Note: viruses are very sensitive to temperature and storage, avoid re-freezing, keep on dry ice during transport and thaw soon before use.) </li>
</ol>
2. Stereotaxic injection
<ol>
 <li>Anesthetize a 6-10 weeks old mouse with isoflurane and place the animal in prone position on a stereotaxic frame (Figure 2A) using the ear bars and the palate support to properly fix the head. The animal is kept warm on a heating pad set at 37 &deg;C and under constant isoflurane administration.</li>
 <li>Inject subcutaneously 100 &mu;l of buprenorphine working solution as pre-emptive analgesic, shave a stripe of skin on the head of the mouse, and desinfect as described in 1.4. Using a scalpel, make a ca. 1.5 cm long longitudinal incision on the head skin exposing the skull at the level of the sagittal suture. Retract the skin and gently clean the bone surface with a sterile cotton bud. </li>
 <li>Half-fill a glass capillary (specifications in 1.2) with paraffin oil and mount it on the injector pump inserting the capillary until the second ring (Figure 2B). Move the capillary holder in the x, y and z axis until the tip of the capillary is positioned on the bregma, the conjunction point between the sagittal and lateral sutures (Figure 2C) (to be determined with the help of a stereomicroscope), and re-set x, y, and z values to 0.</li>
 <li>Move the capillary by &plusmn;1.6 mm in the medio-lateral axis (x) and -1.9 mm in the anterior-posterior axis (y) and mark the bone using a surgical marker pen. Make a hole of ca. 0.5 mm diameter on the skull using an electric driller paying attention not to damage the brain.</li>
 <li>Put a 5 &mu;l droplet of viral suspension (1 aliquot) on a piece of parafilm placed on the ear bars and immerge the tip of the capillary in the droplet. Suck ca. 3.5 &mu;l of viral suspension using a nanoliter injector set at 55 nl/sec. Execute this step rapidly as the droplet will evaporate. </li>
 <li>Move the capillary to the site of injection and move it down until the tip touches the pial surface. Reset the dorso-ventral axis value (z) to 0. At this point, penetrate the tissue with the capillary for -1.9 mm and release 1.5 &mu;l of viral suspension at a rate of 4 nl/sec. Wait for 2-3 minutes to minimize the backflow of viral suspension trough the capillary track and then retract the capillary. A second injection can be performed in the controlateral hemisphere. </li>
 <li>Suture the skin, disinfect, and allow the animal to recover from anesthesia as described previously for in utero electroporation (1.8). </li>
</ol>
<img src="/files/ftp_upload/4093/4093fig2.jpg" alt="Figure 2" /> 
Figure 2. Stereotaxic viral injection. (A and B) Schematic representation of the components needed to perform stereotaxic injection (A) and a disassembled capillary holder showing the insertion of a capillary until the second ring (B). (C) Picture of the mouse head immobilized by the ear bars and palate support with the skin cut to expose the skull (left). The magnification of the dotted box (right) shows the lateral and sagittal sutures, the bregma, and the site of injection (circle).
3. Processing of the sample
<ol>
 <li>One-to-three weeks post-infection, perfuse the mouse with ca. 100 ml of 4% PFA, dissect the brain and post-fix it overnight in 4% PFA. Eventually, BrdU and/or tamoxifen can be administered before sacrifice according to different paradigms to investigate cell cycle kinetics and to stop the expression of the viral transgenes flanked by loxP sites, respectively 9.</li>
 <li>The brain can then be processed for immunostaining using several markers of stem and progenitors cells and neurons (e.g. Sox2, GFAP, Nestin, Tbr2, doublecortin, etc). </li>
</ol>

<ol>
	<li>Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells:+units+of+development,+units+of+regeneration,+and+units+in+evolution.">Stem cells: units of development, units of regeneration, and units in evolution.</a> Cell. 100, 157-168 (2000).</li><li>Lindvall, O., Kokaia, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+for+the+treatment+of+neurological+disorders.">Stem cells for the treatment of neurological disorders.</a> Nature. 441, 1094-1096 (2006).</li><li>Salomoni, P., Calegari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+control+of+mammalian+neural+stem+cells:+putting+a+speed+limit+on+G1.">Cell cycle control of mammalian neural stem cells: putting a speed limit on G1.</a> Trends Cell Biol. 5, 332-342 (2010).</li><li>Lange, C., Calegari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdks+and+cyclins+link+G(1)+length+and+differentiation+of+embryonic,+neural+and+hematopoietic+stem+cells.">Cdks and cyclins link G(1) length and differentiation of embryonic, neural and hematopoietic stem cells.</a> Cell Cycle. 9, 1893-1900 (2010).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev. Biol. 240, 237-246 (2001).</li><li>Inoue, T., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+impulse+to+the+brain--using+in+vivo+electroporation.">An impulse to the brain--using in vivo electroporation.</a> Nat. Neurosci. 4, 1156-1158 (2001).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 10, 865-872 (2001).</li><li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex.">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cereb. Cortex. , 120-125 (2009).</li><li>Artegiani, B., Lindemann, D., Calegari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+cdk4+and+cyclinD1+triggers+greater+expansion+of+neural+stem+cells+in+the+adult+mouse.">Overexpression of cdk4 and cyclinD1 triggers greater expansion of neural stem cells in the adult mouse.</a> J. Exp. Med. 208, 937-948 (2011).</li><li>Dixit, R., Lu, F., Cantrup, R., Gruenig, N., Langevin, L. M., Kurrasch, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Gene+Delivery+into+Multiple+CNS+Territories+Using+In+Utero+Electroporation.">Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation.</a> J. Vis. Exp. (52), e2957 (2011).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Utero+Intraventricular+Injection+and+Electroporation+of+E15+Mouse+Embryos.">In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos.</a> J. Vis. Exp. (6), e239 (2007).</li><li>Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+in+Utero+Electroporation:+Controlled+Spatiotemporal+Gene+Transfection.">Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection.</a> J. Vis. Exp. (54), e3024 (2011).</li><li>Lowery, R. L., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+Injection+of+Adeno-associated+Viral+Vectors.">Intracranial Injection of Adeno-associated Viral Vectors.</a> J. Vis. Exp. (45), e2140 (2010).</li><li>Lepousez, G., Alonso, M., Wagner, S., Gallarda, B. W., Lledo, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Viral+Transduction+of+Adult-born+Olfactory+Neurons+for+Chronic+in+vivo+Optogenetic+Stimulation.">Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation.</a> J. Vis. Exp. (58), e3380 (2011).</li><li>Lange, C., Huttner, W. B., Calegari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdk4/cyclinD1+overexpression+in+neural+stem+cells+shortens+G1,+delays+neurogenesis,+and+promotes+the+generation+and+expansion+of+basal+progenitors.">Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors.</a> Cell Stem Cell. 5, 320-331 (2009).</li><li>De Pietri Tonelli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+detection+of+microRNAs+in+developing+vertebrate+embryos+after+acute+administration+of+a+dual-fluorescence+reporter/sensor+plasmid.">Single-cell detection of microRNAs in developing vertebrate embryos after acute administration of a dual-fluorescence reporter/sensor plasmid.</a> Biotechniques. 41, 727-732 (2006).</li><li>Calegari, F., Marzesco, A. M., Kittler, R., Buchholz, F., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+RNA+interference+in+post-implantation+mouse+embryos+using+directional+electroporation+and+whole+embryo+culture.">Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture.</a> Differentiation. 72, 92-102 (2004).</li><li>Takahashi, M., Sato, K., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+gene+expressions+by+electroporation+in+the+developing+brain+of+mammalian+embryos.">Manipulating gene expressions by electroporation in the developing brain of mammalian embryos.</a> Differentiation. 70, 155-162 (2002).</li><li>Pierfelice, T. J., Gaiano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-Guided+Microinjection+into+the+Mouse+Forebrain+In+Utero+at+E9.5.">Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5.</a> J. Vis. Exp. (45), e2047 (2010).</li><li>Osumi, N., Inoue, T. G. e. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=transfer+into+cultured+mammalian+embryos+by+electroporation.">transfer into cultured mammalian embryos by electroporation.</a> Methods. 24, 35-42 (2001).</li></ol>

BA and FC have submitted a patent application describing the conditional expansion of adult somatic stem cells by cdk4/cyclinD1 viruses (EP 10160288.6).

A critical step for in utero electroporation is the quality and purity of the DNA since its directional migration during delivery of the electric fields depends upon its charge. Protocols for DNA purification that do not include removal of additional charged molecules, such as endotoxins, may lead to the masking of the natural DNA negative charges leading to poor delivery. Clearly, touching the uterine walls with the electrodes as close as possible to the area to be targeted will improve electroporation efficiency. Equally important is the quality of the electrodes used since this is fundamental for the delivery of optimal electric fields. Micro-scratches not even visible by eye and/or organic residues, such as traces of serum, lipids, and so forth, will inevitably accumulate over time leading to electrodes losing their properties. Old and/or dirty electropes are the most likely cause of a sudden drop in electroporation efficiency and, thus, should be replaced as soon as this is noticed. While relatively simple, in utero electroporation typically requires a few weeks of training in order to achieve satisfactorily and reproducible results. This technique is very versatile since it can be applied, virtually, to any organ and tissue of the developing embryo. Certainly, organs containing a cavity, such as the brain, are particularly easy to target and allow for a high proportion of transfected cells due to the ease of injecting a relatively large volume of DNA.
A common problem encountered in setting up stereotaxic viral injection is to have few infected cells and/or to target undesired areas. The first problem is mainly correlated with the injection of low-titer viruses. 293T cells with lower number of passages in culture grow faster and lead to higher viral titers. Using cells older than ca. 20 passages is not recommended and means of transfection with less than 80% efficiency will not ensure a good viral production. Resuspension of the viral pellet is also critical, the viral suspension has to appear homogenous and viral clumps that could occlude the capillary have to be avoided. Viruses are very sensitive to temperature drops and long term storage. For this reasons, aliquots need to be permanently stored at -80 &deg;C and cannot be refrozen. Even under these conditions, a decrease in infectivity could be observed after 6-12 months of storage. In the learning phases, we suggest to test the viral titer of each viral preparation and to do it again after long term storage. The presence of infected cells in undesired areas of the brain can depend on a suboptimal positioning of the mouse head on the stereotaxic frame, which requires some practice. In addition, it is critical not to damage the surface of the brain while opening the hole on the skull because otherwise it will not be possible to reset the 0 correctly on the pial surface, resulting in imprecise injection. The coordinates that we provided in this protocol are referred to C57/BL6 6-10 weeks old females and we suggest optimizing them for different strain/age/sex. Acquisition of this technique may require more time than in utero electroporation due to the longer phases needed to prepare the viruses and analyze the samples. We strongly recommend that viruses are only used after mouse surgery and stereotaxic injection have become reliable and reproducible. The first step to achieve this is to inject cell-permeant DNA dyes, such as Hoechst 33342, in euthanized mice and analyze the brains immediately.
In utero electroporation and stereotaxic viral injection are two powerful systems to acutely and tissue-specifically manipulate gene expression within the CNS during mammalian development and adulthood. Despite their limitation in targeting only a subpopulation of cells, these systems provide an unprecedented speed and ease as compared to more conventional methods, in particular the laborious and time consuming generation of transgenic mice. Yet, despite this similarity, several differences exist between the two techniques that are worth mentioning. First, with regard to the targeting of different cell types, in utero electroporation initially leads to the transfection of stem cells proper (also called radial glial cells) because these are the only cells delimiting the ventricle where the DNA is injected. As a result of cell divisions, plasmids will subsequently be inherited from targeted, mother stem cells to their progenitor and/or neuronal daughter cells leading to the redistribution of transfected cells throughout the cortex as a function of time. In contrast, the use of HIV lentiviruses in the adult brain leads to the simultaneous infection of any cell type including stem cells, progenitors, neurons as well as mature glial cells. Of note, with regard to using HIV lentiviruses as opposed to more commonly used retroviruses is the fact that lentiviral integration would occur independently from the mitotic state of cells, thus allowing the targeting also of adult, quiescent stem cells.
Second, another important difference between electroporation and viral infection is that a transitory, episomal expression is achieved by the former as opposed to the irreversible integration of DNA in the genome of infected cells achieved by the latter. As a result of consecutive cell divisions, dilution of the electroporated plasmids, and degradation of the ectopic cyclinD1 protein, would ensue at each cell cycle. Thus, expansion of NSC during embryonic development was shown to only last for approximately two days 15 while in the adult brain this effect was found to lasts for several months (unpublished results).
Third, since i) cdk4/cyclinD1 has a stronger effect on cells with a longer G1 and ii) committed neural progenitors during development have a longer cell cycle than stem cells while the opposite holds true during adulthood, our two manipulations were found to trigger the expansion, primarily, of neural progenitors during development and stem cells during adulthood. In either case an increase in neurons has been achieved by both methods. 
Fourth, viral injection can easily be performed, reliably and reproducibly, in many different regions of the adult brain by simply changing the stereotaxic coordinates. In contrast, precise targeting of certain regions of the central nervous system, such as brain stem, hippocampal formation, or spinal cord, by in utero electroporation might be more difficult to achieve reproducibly. Moreover, while stereotaxic injection can be performed on mice of any age, optimal performance of in utero electroporation is limited from about E11 to E16. Stages prior to E11 would require either the use of an ultrasound backscatter microscope 18,19 or a whole embryo culture system 17,20.
Recently, an increasing interest in basic cell biology of somatic stem cells has been promoted because their study and manipulation are thought to be fundamental towards developing novel cell-based regenerative therapies. In particular for overexpression of cdk4/cyclinD1, our protocol provides the means to transitorily increase the expansion of NSC in vivo (Figure 3B and C and 4B and C) in order to increase the number of neurons generated in the adult brain. We believe that these manipulation may be important in a number of different contexts to better understand the role of NSC in brain development, evolution and homeostasis as well as their contribution to cognitive function or recovery from neural degeneration or injury.

<table border="1">
<tbody>
 <tr>
 <td>Material/equipment </td>
 <td>Supplier </td>
 <td>Catalog # </td>
 <td></td>
 </tr>
 <tr>
 <td>Endofree maxi plasmid kit</td>
 <td>Qiagen</td>
 <td>12362</td>
 <td></td>
 </tr>
 <tr>
 <td>FastGreen</td>
 <td>Sigma</td>
 <td>F7258</td>
 <td></td>
 </tr>
 <tr>
 <td>P-97 Flaming/Brown pipette puller</td>
 <td>Sutter Instruments</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Borosilicate capillaries with filament</td>
 <td>Sutter Instruments</td>
 <td>BF120-69-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Table top laboratory animal anesthesia system</td>
 <td>VetEquip</td>
 <td>901806</td>
 <td></td>
 </tr>
 <tr>
 <td>Isofluran </td>
 <td>Baxter </td>
 <td>HDG9623 </td>
 <td></td>
 </tr>
 <tr>
 <td>Germinator 500 glass bead sterilizer</td>
 <td>SouthPointe Surgical</td>
 <td>GRM5-1460</td>
 <td></td>
 </tr>
 <tr>
 <td>Iris forceps</td>
 <td>WPI</td>
 <td>15917</td>
 <td></td>
 </tr>
 <tr>
 <td>Betaisodona solution</td>
 <td>Mundipharma</td>
 <td>1931491</td>
 <td></td>
 </tr>
 <tr>
 <td>Pico Pump </td>
 <td>WPI</td>
 <td>PV820</td>
 <td></td>
 </tr>
 <tr>
 <td>ECM 830 square wave electroporator</td>
 <td>Harvard Apparatus</td>
 <td>450052</td>
 <td></td>
 </tr>
 <tr>
 <td>Round tweezer electrodes</td>
 <td>NEPAGENE</td>
 <td>CUY650P1</td>
 <td></td>
 </tr>
 <tr>
 <td>Suture material</td>
 <td>Ethicon</td>
 <td>V493H</td>
 <td></td>
 </tr>
 <tr>
 <td>Reflex clip applier for wound closure</td>
 <td>WPI</td>
 <td>500345</td>
 <td></td>
 </tr>
 <tr>
 <td>Temgesic injection solution</td>
 <td>Essex Pharma AG</td>
 </tr>
 <tr>
 <td>DMEM w/Glutamax-I</td>
 <td>Invitrogen </td>
 <td>31966021</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal Bovin Serum </td>
 <td>Invitrogen </td>
 <td>1027016</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin/Streptomicin </td>
 <td>Gibco </td>
 <td>15140</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue culture dish</td>
 <td>Falcon</td>
 <td>353003</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyethylenimine 250 ml </td>
 <td>Sigma-Aldrich</td>
 <td>40872-7</td>
 <td></td>
 </tr>
 <tr>
 <td>n-Butyric acid, sodium salt</td>
 <td>Sigma</td>
 <td>B 5887</td>
 <td></td>
 </tr>
 <tr>
 <td>Tube, thinwall, polyallomer </td>
 <td>Beckman</td>
 <td>326823 / 331374</td>
 <td></td>
 </tr>
 <tr>
 <td>Model 900 small animal stereotaxic instrument</td>
 <td>Kopf Instruments</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>OPMI pico microscope</td>
 <td>Zeiss</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>SYS-Micro4 controller and Nanoliter2000 injector</td>
 <td>WPI</td>
 <td>B203MC4</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass replacement 3.5 nanoliter</td>
 <td>WPI</td>
 <td>4878</td>
 <td></td>
 </tr>
 <tr>
 <td>SR drill general application</td>
 <td>Foredam </td>
 <td>K2272 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>
 <table border="1">
 <tbody>
 <tr>
 <td>Antibody&nbsp; </td>
 <td>Supplier </td>
 <td>Catalog # </td>
 <td>Dilution</td>
 </tr>
 <tr>
 <td>Doublecortin</td>
 <td>Santacruz</td>
 <td>SC8066</td>
 <td>1:100</td>
 </tr>
 <tr>
 <td>GFAP</td>
 <td>Chemicon</td>
 <td>MAB3404</td>
 <td>1:500</td>
 </tr>
 <tr>
 <td>Nestin</td>
 <td>BD Biosciences</td>
 <td>556309</td>
 <td>1:1000</td>
 </tr>
 <tr>
 <td>S100&beta; </td>
 <td>Abcam</td>
 <td>Ab868</td>
 <td>1:1000</td>
 </tr>
 <tr>
 <td>Sox2</td>
 <td>Millipore</td>
 <td>AB-5603</td>
 <td>1:500</td>
 </tr>
 <tr>
 <td>Tbr2 </td>
 <td>Abcam </td>
 <td>Ab23345 </td>
 <td>1:400</td>
 </tr>
 <tr>
 <td colspan="4">Table of antibodies.</td>
 </tr>
 </tbody>
 </table>
 </td>
 </tr>
 </tbody>
 </table>

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.

<ol>
 <li>Overall, in utero electroporation yields 60-80% of electroporated embryos displaying strong expression of the reporter gene in a wide area of the lateral cortex (Figure 3A). Fluorescent proteins can be easily detected on whole mount embryos as soon as 3-6 hours after surgery with the signal lasting for more than one week. Within the targeted area, about 30-50% of cells usually express the transgene(s) with the proportion of cells co-expressing two (or more) co-electroporated transgenes being usually above 90% (Figure 3B, left) 6,8,15. While a minimal level of apoptosis (ca. 2.0-2.5%) could be observed after about 2 hours from electroporation, this value will return to physiological levels (ca. 0.5-1.0%) after about 6 hours (FC, unpublished data). The proportion of embryos or pregnant mice dying due to the surgery is typically negligible (&lt;5%). Co-electroporation with cdk4/cyclinD1 under these conditions followed by 48 hours of development triggers an increase in the expansion of neural progenitor by 30% (Figure 3B, right, and C) 15.</li>
 </ol>
<img src="/files/ftp_upload/4093/4093fig3.jpg" alt="Figure 3" /> 
Figure 3. Expansion of neural progenitors by cdk4/cyclinD1 overexpression. (A) Fluorescence picture of a section through the lateral cortex of a mouse embryo 24 hours after electroporation with GFP and RFP plasmids at embryonic day 13. (B) Fluorescent pictures as in A 48 hours after co-electroporation with cdk4/GFP and cyclinD1/RFP plasmids and immunolabeling for the basal progenitor marker Tbr2. Fluorescent reporters with DAPI counterstainig (left) and Tbr2 (right) are shown. Lines indicates the apical surface of the ventricular zone (VZ; white line) and the boundaries of the subventricular zone (SVZ; yellow dashed lines). Note the increased thickness of the SVZ in the electroporated portion of the cortex (white arrowheads) due to an increased generation and expansion of basal progenitors after overexpression of cdk4/cyclinD1. (C) Quantification of the effect shown in B. Bars=SD; p&lt;0.05; n=3. Bar graph in C is taken from Lange et al., 2009 15. 
<ol start="2">
 <li>After viral stereotaxic injection, ca. 80% of operated mice display strong expression of the transgenes throughout the whole dentate gyrus. Constitutive expression of GFP can be easily detected on 40 &mu;m sections as soon as 4 days after surgery. Along the rostral-to-caudal axis of the dentate gyrus, about 10-30% of cells (equivalent to a total of ca. 10,000-30,000 cells) usually express the transgene(s) (Figure 4A) 9. The proportion of mice dying due to the surgery is negligible (&lt;5%). Three week overexpression of cdk4/cyclinD1 under these conditions triggers an increase in NSC by over two-folds (Figure 4B) while decreasing neurogenesis by 70% (Figure 4C) 9.</li>
</ol>
<img src="/files/ftp_upload/4093/4093fig4.jpg" alt="Figure 4" /> 
Figure 4. Effects of viral infection. (A) 3D reconstruction of 7 fluorescence pictures taken from 40 &mu;m thick serial coronal sections (collecting 1 every 6) along the rostral-to-caudal axis of the hippocampus. GFP+ infected cells (green) and DAPI nuclear staining (blue) of the dentate gyrus are shown (top). Bright field pictures of the coronal sections corresponding to those shown in A (middle) and 3D representation (bottom) of the whole cerebral hemisphere with the hippocampus highlighted in green pseudocolor (modified from the Allen Brain Atlas; <a href="http://www.brain-map.org" target="_blank">www.brain-map.org</a>). White arrows point the site of injection. Lateral (L), rostral (R), and dorsal (D) axes (x, y, and z stereotaxic coordinates) are indicated (right). (B and C) Proportion of nestin+ NSC (B) and DCX+ newborn neurons (C) within the population of GFP+ infected cells 3 weeks after stereotaxic injection of GFP (white) or cdk4/cyclinD1/GFP (black) viruses. Bars=SD; p&lt;0.005; n=3. Graphs in B and C taken from Artegiani et al., 2011.

Watch this Scientific Journal Video about Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection at JoVE.com

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.

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 ...

expansion-embryonic-adult-neural-stem-cells-utero-electroporation-or

Research

JoVE Journal

Neuroscience

16.7K Views.  DFG - Research Center and Cluster of Excellence for Regenerative Therapies Dresden, Germany. 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.

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

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

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 ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo.

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Isolation and Culture of Embryonic Mouse Neural Stem Cells

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation