This work was supported by grants from the Deutsche Forschungsgemeinschaft to SB (BR-2215; SFB 497/A9).

NOTE: All animal experiments were carried out in accordance with the German law and were approved by the government offices in T&#252;bingen.
1. Preparation of Micropipettes, Solutions and Membranes
<ol>
	<li>Preparation of Micropipettes
	<ol>
		<li>Pull glass micropipettes using a micropipette puller with the following program: Heat: 540, Pull: 125, Velocity: 20 and Delay: 140. The needle length amounts to 5.5 cm.</li>
		<li>Bevel needles using a microgrinder to obtain a suitable tip size of 4 mm. Store the needles in a box or 15 cm Petri dish to prevent damaging of the tips.</li>
	</ol>
	</li>
	<li>Preparation of Solutions
	<ol>
		<li>Plasmid DNA Solution
		<ol>
			<li>Prepare plasmid DNA containing the desired cDNA construct using an Endotoxin free Maxi-prep kit according to manufacturer&#8217;s protocol.</li>
			<li>Adjust plasmid DNA solution to a final concentration of 3 &#181;g/&#181;l (without GFP spike vector) or 4 &#181;g/&#181;l (with 1 &#181;g of GFP spike vector) in endotoxin free Tris-EDTA buffer containing Fast Green (final concentration 0.05%).</li>
		</ol>
		</li>
		<li>Laminin Stock solution
		<ol>
			<li>Dissolve 1 mg of laminin in sterile water to a final volume of 1 ml. Prepare 100 &#181;l aliquots and store at -80 &#176;C.</li>
		</ol>
		</li>
		<li>Poly-L-Lysine Stock Solution
		<ol>
			<li>Dissolve 50 mg of poly-L-lysine in 50 ml of sterile water to a final concentration of 1 mg/ml. Prepare 1 ml aliquots and store in -20 &#176;C.</li>
		</ol>
		</li>
		<li>Complete Hank&#8217;s Balanced Salt Solution (Complete HBSS)
		<ol>
			<li>Prepare Complete HBSS by combining 100 ml of 10x HBSS, 2.5 ml of 1 M HEPES buffer (pH 7.4), 30 ml of 1 M D-glucose, 10 ml of 100 mM CaCl2, 10 ml of 100 mM MgSO4, and 4 ml of 1 M NaHCO3. Add sterile water up to 1 L and store at 4 &#176;C. 
			NOTE: Autoclave all solutions expect 1 M HEPES buffer and 1 M D-glucose, which are filter sterilized.</li>
		</ol>
		</li>
		<li>Slice Culture Medium
		<ol>
			<li>Prepare slice culture medium by adding 35 ml of Basal Medium Eagle, 12.9 ml of complete HBSS (1.2.4), 1.35 ml of 1 M D-glucose, 250 &#181;l of 200 mM L-glutamine, and 500 &#181;l of penicillin-streptomycin to obtain a final volume of 50 ml. Add horse serum to a final concentration of 5% and store at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Low-Melting Point (LMP) Agarose
		<ol>
			<li>Prepare a 4% LMP agarose solution by adding 2 g of LMP agarose to 50 ml of complete HBSS (1.2.4) followed by heating in a microwave for 1-2 min at high power. Keep this solution in a water bath at 37 &#8211; 39 &#176;C. Store the solution at 4 &#176;C and reuse.</li>
		</ol>
		</li>
		<li>Paraformaldehyde Solution
		<ol>
			<li>In a fume hood prepare a 4% paraformaldehyde (PFA) solution by adding 4 g of paraformaldehyde to 100 ml of 1x PBS. Heat the solution to 60 &#176;C and add a few drops of 1 N NaOH until the solution becomes clear.</li>
		</ol>
		</li>
		<li>Permeabilization Solution
		<ol>
			<li>Dissolve 9 g of BSA in 300 ml of 1x PBS containing 0.3% Trition X-100 and store at 4 &#176;C. Add 10% sodium azide for long term storage.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Coating of Membrane Inserts
	<ol>
		<li>Dilute one aliquot of laminin stock solution (1.2.2) and one aliquot of poly-L-lysine stock solution (1.2.3) in sterile water to a final volume of 12 ml.</li>
		<li>Place membrane inserts into 6 well plates with each well containing 2 ml of sterile water. Add 1 ml of coating solution on top of the membrane and incubate O/N at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>After incubation wash the membrane inserts three times with 1 ml of sterile water and dry. Use coated membrane inserts on the same day or store at 4 &#176;C for up to four weeks in a dry 6 well plate.</li>
	</ol>
	</li>
</ol>
2. DNA Injection and Electroporation of E15.5 and E18.5 Embryos
<ol>
	<li>Anesthetize a time mated female mouse by placing it into an anesthetizing chamber saturated with 5% isoflurane and connected to a vaporizer. Circulate isoflurane and oxygen at a rate of 1 L/min. Keep the animal in the box for 2-4 min or until unconscious which is tested by pinching between the paws of the mouse.</li>
	<li>Euthanize the unconscious mouse by cervical dislocation on embryonic day (E) 15.5 or 18.5. Dissect the uterus containing the embryos13 and place it into a Petri dish containing 15-20 ml of cold complete HBSS. 
	NOTE: From this point onwards, keep the embryos and tissues on ice.</li>
	<li>Use a pair of scissors to separate each embryo from the uterine horn and place into a second Petri dish containing cold complete HBSS.</li>
	<li>Under a dissecting microscope, sever the uterine muscle wall and the placenta using a pair of fine forceps (#55) and scissors. Carefully release the embryo from the yolk sac.</li>
	<li>Use a pair of Bonn scissors to decapitate the embryos just above the forelimbs at a 60&#176; angle. If the experiment requires genotyping of the embryos, collect a tissue sample for genomic DNA isolation (a small piece of tail).</li>
	<li>Transfer the head to a clean and dry Petri dish. Because the head had been decapitated in a 60&#176; angle, the head should tilt to one side when placed dorsal side up.</li>
	<li>Place a needle carefully into the middle of the hemisphere close to the bregma (Figure 1A, B). Inject approximately 2-3 &#181;l (at 3 or 4 &#181;g/&#181;l) of DNA solution by stepping on the pedal of the picospritzer III using 30 pounds of pressure for the duration of 10-15 msec per pulse, applying 5-8 pulses. The duration and number of the pulses depends on the diameter of the needle opening with smaller openings requiring more time. The interval between each pulse amounts to 1 sec.</li>
	<li>Before placing the electrodes, apply a few drops of complete HBSS on the head of the embryo. Place the electrodes in such a way that the &#8216;negative&#8217; terminal is on the same side as the injected ventricle and the &#8216;positive&#8217; electrode on the opposite side of the injected ventricle below the ear of the embryo&#8217;s head (Figure 1C, D). Apply 5 pulses of 50 V.
	<ol>
		<li>Use 3 mm electrodes for E 15.5 and 5 mm electrodes for E 18.5.</li>
	</ol>
	</li>
</ol>
3. Dissection of the Brain
<ol>
	<li>After the electroporation, peel off the skin from the head with the help of a pair of fine forceps. Using a pair of spring scissors make a small incision in the middle of the cerebellum at the midline of the skull.</li>
	<li>Insert the spring scissors into the incision and cut longitudinally along the sagittal suture. Peel off the skull and detach the brain from the skull by using fine forceps. Transfer the whole brain into 15-20 ml cold complete HBSS solution.</li>
	<li>Meanwhile, pour 4% LMP agarose, kept at 37-39 &#176;C in a water bath, into a peel-away mold.</li>
	<li>Take the brain out of the complete HBSS by a small scoop spatula and drain excess of HBSS by using fine tissue paper or Kimwipes.</li>
	<li>Place the whole brain gently into the agarose and adjust its position with a fine needle. Keep the mold on ice until the agarose is solidified and the block is sectioned (for coronal sections the olfactory bulbs point up).</li>
</ol>
4. Vibratome Sectioning and Slice Culture
<ol>
	<li>Trim the LMP agarose blocks and glue them to the specimen stage using &#8216;super glue&#8217;. After the glue is dry transfer the specimen stage to the buffer tray of the vibratome and fill with ice cold complete HBSS until the block is immersed in the solution. 
	NOTE: Sterilize all instrument and equipment surfaces with 70% ethanol before sectioning.</li>
	<li>Prepare 250 &#181;m thick vibratome sections using a new blade as followed.
	<ol>
		<li>Trim the block with the following settings; frequency &#8211; 60 Hz, amplitude &#8211; 0.7 &#181;m, speed 16-18 mm/sec. Cut the sections containing the desired tissue with the above settings at slow speed (9 mm/sec). Starting the sectioning from the hindbrain, collect 5-7 sections from the cerebrum.</li>
		<li>Transfer the sections to a clean 6 well culture dish containing 5 ml of ice cold complete HBSS with the help of a bent spatula and keep on ice till all the sections are collected (Figure 1E).</li>
	</ol>
	</li>
	<li>Moisten the membrane in a cross fashion with 100 &#181;l of complete HBSS before placing the sections on the membrane, to facilitate the orientation of the sections.</li>
	<li>Transfer the sections using a bent spatula onto the membrane (pick up a corner of the section with forceps and pull onto the spatula and then use the forceps to push the section onto the membrane). Place up to five sections on one membrane and arrange by using forceps (Figure 1F). Do not overlap the sections with each other.
	<ol>
		<li>Take the excess of HBSS off the membrane using a pipette. The specific membranes used here are attached to a frame and inserted into the tissue culture plate, which allows the tissue to be in contact but not covered by the medium.</li>
	</ol>
	</li>
	<li>Place the membrane inserts into a 6 well plate containing 1.8 ml of slice culture medium (1.2.5) (Figure 1G, H). Incubate the culture dish at 37 &#176;C with 5% CO2 for 11 DIV or 14 DIV. Change half the medium (0.9 ml) every second day. 
	NOTE: At this stage, add reagents like Bromodeoxyuridine (BrdU; 10 &#181;M final concentration) for labeling proliferating cells to the media for the first 20 hr of the culture time.</li>
</ol>
5. Fixation of the Sections Followed by Immunofluorescence Staining
<ol>
	<li>Use a clean and sharp scalpel blade to cut and trim the membranes depending on the orientation of the sections.</li>
	<li>Transfer the sections along with the membrane to a 24 well plate containing 1 ml of 4% PFA (1.2.7). Incubate the sections for 1 hr at RT followed by 3 washes with 1x PBS for 15 min each. Incubate the sections O/N with permeabilization solution at 4 &#176;C with gentle agitation.</li>
	<li>The following day, incubate the sections with appropriate primary antibodies, diluted in permeabilization solution, O/N or for 48 hr at 4 &#176;C with gentle agitation.</li>
	<li>Wash the sections 3 times for 15 min with 1x PBS and incubate O/N at 4 &#176;C with the appropriate secondary antibodies diluted in permeabilization solution.</li>
	<li>After the incubation with secondary antibodies, wash the sections once with 1x PBS for 15 min followed by DAPI staining for 10 min.</li>
	<li>Wash the sections 3 times with 1x PBS for 15 min each and transfer to microscope slides. Add ImmunoMount and gently place a coverslip on top of the sections. Dry the slides O/N at 4 &#176;C and seal with nail polish. 
	NOTE: Keep the slides always at 4 &#176;C.
	<ol>
		<li>Analyze the slice cultures by confocal microscopy (Figure 1I).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kempermann, G., Jessberger, S., Steiner, B., Kronenberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milestones+of+neuronal+development+in+the+adult+hippocampus.">Milestones of neuronal development in the adult hippocampus.</a> Trends Neurosci. 27, 447-452 (2004).</li><li>Frotscher, M., Zhao, S., Forster, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+cell+and+fiber+layers+in+the+dentate+gyrus.">Development of cell and fiber layers in the dentate gyrus.</a> Prog Brain Res. 163, 133-142 (2007).</li><li>Muramatsu, R., Ikegaya, Y., Matsuki, N., Koyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatally+born+granule+cells+numerically+dominate+adult+mice+dentate+gyrus.">Neonatally born granule cells numerically dominate adult mice dentate gyrus.</a> Neuroscience. 148, 593-598 (2007).</li><li>Li, G., Pleasure, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+the+dentate+gyrus:+what+we+are+learning+from+mouse+mutants.">Morphogenesis of the dentate gyrus: what we are learning from mouse mutants.</a> Dev Neurosci. 27, 93-99 (2005).</li><li>Hsieh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orchestrating+transcriptional+control+of+adult+neurogenesis.">Orchestrating transcriptional control of adult neurogenesis.</a> Genes Dev. 26, 1010-1021 (2012).</li><li>Li, G., Pleasure, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+dentate+gyrus+morphogenesis.">Genetic regulation of dentate gyrus morphogenesis.</a> Prog Brain Res. 163, 143-152 (2007).</li><li>Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+state+of+chromatin+immunoprecipitation.">The current state of chromatin immunoprecipitation.</a> Mol Biotechnol. 45, 87-100 (2010).</li><li>Simon, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+function+of+Bcl11b/Ctip2+in+hippocampal+neurogenesis.">A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis.</a> Embo J. 31, 2922-2936 (2012).</li><li>Pilaz, L. J., Silver, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+mitosis+in+the+developing+mouse+embryonic+cortex.">Live imaging of mitosis in the developing mouse embryonic cortex.</a> J Vis Exp. (88), (2014).</li><li>Pacary, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+genetic+manipulation+of+dendrites+and+spines+in+the+mouse+cerebral+cortex+and+hippocampus+using+in+utero+electroporation.">Visualization and genetic manipulation of dendrites and spines in the mouse cerebral cortex and hippocampus using in utero electroporation.</a> J Vis Exp. (65), (2012).</li><li>Hand, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Neurogenin2+specifies+the+migration+properties+and+the+dendritic+morphology+of+pyramidal+neurons+in+the+neocortex.">Phosphorylation of Neurogenin2 specifies the migration properties and the dendritic morphology of pyramidal neurons in the neocortex.</a> Neuron. 48, 45-62 (2005).</li><li>Polleux, F., Ghosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+slice+overlay+assay:+a+versatile+tool+to+study+the+influence+of+extracellular+signals+on+neuronal+development.">The slice overlay assay: a versatile tool to study the influence of extracellular signals on neuronal development.</a> Sci STKE. (136), 19 (2002).</li><li>Shea, K., Geijsen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+6.5+dpc+mouse+embryos.">Dissection of 6.5 dpc mouse embryos.</a> J Vis Exp. (2), (2007).</li><li>Sugiyama, T., Osumi, N., Katsuyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+germinal+matrices+in+the+developing+dentate+gyrus+are+composed+of+neuronal+progenitors+at+distinct+differentiation+stages.">The germinal matrices in the developing dentate gyrus are composed of neuronal progenitors at distinct differentiation stages.</a> Dev Dyn. 242, 1442-1453 (2013).</li><li>Lechler, T., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desmoplakin:+an+unexpected+regulator+of+microtubule+organization+in+the+epidermis.">Desmoplakin: an unexpected regulator of microtubule organization in the epidermis.</a> J Cell Biol. 176, 147-154 (2007).</li><li>Nichols, A. J., O'Dell, R. S., Powrozek, T. A., Olson, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+utero+electroporation+and+whole+hemisphere+explants:+a+simple+experimental+method+for+studies+of+early+cortical+development.">Ex utero electroporation and whole hemisphere explants: a simple experimental method for studies of early cortical development.</a> J Vis Exp. (74), (2013).</li></ol>

The authors declare that they have no competing financial interests.

The hippocampus has an important function in learning and memory. The dentate gyrus is also one of two brain regions where neurogenesis occurs not only during development but also throughout adulthood. Postnatal and adult hippocampal neurogenesis proceeds in a similar way involving many common factors. Defining the regulatory mechanisms of these factors will be very helpful in understanding neurodegenerative diseases which in turn will lead to new therapies and preventive measures. To obtain this information one requires...

The hippocampus plays an important role in memory and learning as well as emotional behavior. One main function consists of the consolidation of short-term memory into long-term memory, which requires high plasticity of the nervous system. The dentate gyrus of the hippocampus acts as the primary gateway for input information and is also one of two brain regions with ongoing neurogenesis throughout adulthood1,2. The development of the hippocampal structure occurs during late embryogenesis and particularly during the first 3 to 4 weeks postnatal3. During early development of the dentate gyrus a stem cell pool is established required for postnatal as well as adult neurogenesis4. Developing neurons pass through various stages, from the stem cell through several stages of progenitor cells to the immature and finally the mature neuron during postnatal as well as adult neurogenesis. At different stages of neurogenesis the expression of specific genes is required to allow the maturation and integration of new neurons into the hippocampal circuitry5,6.
Using mouse genetics and phenotype analysis by immunohistochemistry as well as molecular methods allowed defining the expression pattern and function of many of these genes. In addition microarray analysis as well as chromatin immunoprecipitation (ChIP) provided information about potential direct and indirect target genes7,8. However, there are still many open questions concerning the regulatory mechanisms of hippocampal development, in particular the development of the dentate gyrus. To gain further insight how specific genes are regulated a system is required allowing the manipulation of a small number of cells by down- or up-regulation of the gene of interest and/or its target genes and follow their fate during development. In utero electroporation of shRNAs, cDNA of genes of interest or Cre recombinase provides such a tool. To ensure the presence of the desired DNA or small RNAs expression plasmids should be used for electroporation. This approach is very successfully implemented in studying cortical development9,10, but is a more challenging approach examining the development of the dentate gyrus due to the position of the hippocampal structures in deeper brain layers.
Ex utero electroporation followed by organotypic slice culture is one approach to circumvent this problem11,12. In contrast to in utero electroporation not the whole embryo but only the head is used allowing therefore to place the electrodes in a more favorable way to direct the shRNA/DNA towards the hippocampus and dentate gyrus. Our group successfully employed ex utero electroporation to study the role of the transcription factor Bcl11b during dentate gyrus development8. Bcl11b has a dual role in dentate gyrus development by regulating progenitor cell proliferation as well as differentiation as was demonstrated by immunohistochemistry. To further define a mechanism for Bcl11b involvement in these processes, protocols of the Polleux group11,12 were adjusted to study the dentate gyrus as described below in the protocol section. In a first approach the question was addressed whether Bcl11b is regulating neuronal cell differentiation cell autonomously. A second approach examined whether Desmoplakin, a direct target gene of Bcl11b, is sufficient to rescue the Bcl11b phenotype.

<table border="0" cellpadding="0" cellspacing="0" width="271">
	<tbody>
		<tr height="106">
			<td height="106" style="height:106px;width:71px;">Name of Reagent/ Equipment</td>
			<td style="width:73px;">Company</td>
			<td style="width:63px;">Catalog Number</td>
			<td style="width:64px;">Comments/ Description</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:71px;">Flaming/ Brown Micropipette Puller</td>
			<td style="width:73px;">Sutter Instruments Company (USA)</td>
			<td style="width:63px;">P-97</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Fine Glass Pipettes</td>
			<td style="width:73px;">Warner Instruments</td>
			<td style="width:63px;">G100F-4</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:71px;">Microgrinder</td>
			<td style="width:73px;">Narishige, Japan</td>
			<td style="width:63px;">EG-44</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:71px;">Anesthetic Bracket unit</td>
			<td style="width:73px;">Harvard Apparatus</td>
			<td style="width:63px;">PY2 34-0412</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Halovet Vaporizer</td>
			<td style="width:73px;">Harvard Apparatus</td>
			<td style="width:63px;">PY2 34-0398</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Fluovac System</td>
			<td style="width:73px;">Harvard Apparatus</td>
			<td style="width:63px;">PY2 34-0387</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">IMS Fluosorber</td>
			<td style="width:73px;">Harvard Apparatus</td>
			<td style="width:63px;">PY2 34-0415</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Anesthetizing Chamber</td>
			<td style="width:73px;">Harvard Apparatus</td>
			<td style="width:63px;">PY2 34-0460</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Electroporator</td>
			<td style="width:73px;">BEX Company</td>
			<td style="width:63px;">CUY21 EDIT</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:71px;">Tweezers with disk electrodes</td>
			<td style="width:73px;">BEX Company</td>
			<td style="width:63px;">LF650P3</td>
			<td style="width:64px;">3 mm electrodes for E15.5</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:71px;">Tweezers with disk electrodes</td>
			<td style="width:73px;">BEX Company</td>
			<td style="width:63px;">LF650P5</td>
			<td style="width:64px;">5 mm electrodes for E18.5</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:71px;">Picospritzer III</td>
			<td style="width:73px;">Parker Hannifin Corporation</td>
			<td style="width:63px;">P/N 052-0500-900</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:71px;">HM 650V Vibrating Blade Microtome, 230V</td>
			<td style="width:73px;">Thermo Scientific</td>
			<td style="width:63px;">920120</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:71px;">Dissection Microscope</td>
			<td style="width:73px;">Carl Zeiss Microscopy Gmbh</td>
			<td style="width:63px;">Stemi SV8</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Inverted Microscope</td>
			<td style="width:73px;">Leica</td>
			<td style="width:63px;">Leica DM IL LED</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Confocal Microscope</td>
			<td style="width:73px;">Leica</td>
			<td style="width:63px;">Sp5II</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:71px;">6 well dish</td>
			<td rowspan="2" style="width:73px;">BD Falcon</td>
			<td rowspan="2" style="width:63px;">#353502</td>
			<td rowspan="2" style="width:64px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:71px;">6 well dish</td>
			<td style="width:73px;">CELLSTAR</td>
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ex utero electroporation and organotypic slice culture of mouse hippocampal tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ablation of the transcription factor Bcl11b causes the impairment of progenitor cell proliferation and neuronal differentiation resulting in a reduced dentate gyrus size and cell number. Furthermore mutant neurons fail to integrate into the hippocampal circuitry causing learning and memory impairment8. To answer questions concerning the regulatory mechanism(s) of Bcl11b in these processes ex utero electroporation was employed.
Addressing the question whether Bcl11b cell-aut...

Watch this Scientific Journal Video about Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue at JoVE.com

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

ex-utero-electroporation-organotypic-slice-culture-mouse-hippocampal

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Developmental Biology

9.4K Views.  University of Ulm. Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Video: Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.