Name: inducing cre-lox recombination in mouse cerebral cortex through in utero electroporation
Uploaded: 2017-11-17T15:00:00
Description: Watch this Scientific Journal Video about Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation at JoVE.com

The authors thank the generous support of the James Madison University Department of Biology and the James Madison University Light Microscopy and Imaging Facility. Dr. Mark L. Gabriele for helpful advice regarding young postnatal tissue preparation, and Drs. Justin W. Brown and Corey L. Cleland for generous coordination of surgical materials and space. This research was funded in part by a Collaborative Research Grant by 4-VA, a collaborative partnership for advancing the Commonwealth of Virginia (G.S.V.), and by a Virginia Academy of Science Small Project Research Grant (G.S.V.). Support has been generously provided by a Betty Jo Loving Butler &#39;58 Endowment for Undergraduate Research Scholarship (to K.M.B.), a Farrell Summer Research Scholarship (to K.M.B.), a James Madison University Second Century Scholarship (to K.M.B.), a James Madison University Centennial Scholarship (to C.J.H.), a James Madison University Lucy Robinson Search &#39;30 Memorial Scholarship (to Z.L.H.), and a James Madison University College of Science and Mathematics Faculty Assistance Grant (to G.S.V.).

Methods described here have been approved by the Animal Care and Use Committee (ACUC) of James Madison University, and are in accordance and compliance with all relevant regulatory and institutional guidelines.
1. Mouse Set-up
<ol>
	<li>House a young (&#62;P60) male and female homozygous floxed mouse together to set up a breeder pair25. 
	NOTE: A good negative control is to set up an additional breeder pair of wildtype mice, in which Cre recombinase expression wi...

<ol>
	<li>Southwell, D. G., Froemke, R. C., Alvarez-Buylla, A., Stryker, M. P., Gandhi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+plasticity+induced+by+inhibitory+neuron+transplantation.">Cortical plasticity induced by inhibitory neuron transplantation.</a> Science. 327 (5969), 1145-1148 (2010).</li><li>Hanson, J. E., Madison, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+FMR1+genotype+influences+the+degree+of+synaptic+connectivity+in+a+mosaic+mouse+model+of+fragile+X+syndrome.">Presynaptic FMR1 genotype influences the degree of synaptic connectivity in a mosaic mouse model of fragile X syndrome.</a> J. Neurosci. 27 (15), 4014-4018 (2007).</li><li>Patel, A. B., Hays, S. A., Bureau, I., Huber, K. M., Gibson, J. 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Here, we introduce the combination of in utero electroporation with Cre recombinase in floxed mice to generate mosaic brain tissue. An advantage of this approach is that a new mouse line does not need to be generated each time a different cellular subtype is to be targeted: in utero electroporation can be used to target excitatory neurons, inhibitory neurons, or glia depending on the time and location of electroporation15,16,<sup clas...

Generating a genetic mosaic is a classic experimental paradigm for understanding the function of a gene of interest. To determine if a gene is necessary for a cellular phenotype, the simplest approach is causing a loss of function of the gene throughout the organism (e.g. knockout). However, to determine if a gene is required specifically in a certain cell type, knockout of the gene throughout the organism is not a valid approach. Instead, a method is required that will cause the loss of function of a gene in a given cell while it is surrounded by wildtype (i.e. genetically unperturbed) tissue&#8212;in other words, creating mosaic tissue. If the mutant cell shows a mutant phenotype, but surrounding wildtype cells do not, the gene functions in a cell-autonomous manner. Analysis of mosaic tissue, in which mutant cells are surrounded by wildtype tissue, is ideal for understanding cell-autonomous functions of genes, especially in the brain where neurons and glia form a vast interconnected network of tissue.
Several forms of mosaic brain tissue have provided powerful models to investigate cell-autonomous functions of genes. Studies focused on neuronal transplantation1, female X-linked mosaicism2,3,4, and endogenous somatic mosaicism5,6 have drawn their conclusions based on mosaic brain tissue. Conditional deletion of a gene through the Cre-lox recombination system is a method that takes full advantage of the great availability of transgenic mouse lines. In this method, two loxP sites are introduced on either side of a required sequence of a gene (such as an exon), leaving it flanked by loxP sites that both face in the same direction (&#34;floxed&#34;). Cre recombinase excises the sequence between the loxP sites7. Cre-mediated recombination can be achieved by crossing floxed mice to another mouse line expressing Cre recombinase along with a fluorescent marker in a subset of cells (&#34;Cre reporter line&#34;). This has been demonstrated in a variety of ways to uncover the functions of a gene in subsets of cells, such as excitatory neurons or astrocytes8. Cre reporter lines can express CreERT2 to allow Cre-mediated recombination to be drug-inducible (single-neuron labeling with inducible Cre-mediated knockout, or SLICK)9. In another strategy called mosaic analysis with double markers (MADM)10,11, Cre-mediated interchromosomal recombination allows a homozygous mutant to be created alongside heterozygous tissue. In these approaches, a new line of mice needs to be produced each time for each candidate gene or cellular subtype that is tested. Alternatively, Cre recombinase can be introduced postnatally through iontophoresis12 or through viral vectors (e.g. adeno-associated viruses13 or lentiviruses14 carrying cellular subtype-specific promoters). This strategy creates strong and postnatal labeling. To target developing cerebral cortical neurons sparsely and prenatally, an ideal strategy is in utero electroporation of Cre recombinase with a fluorescent marker.
In addition to combining Cre-lox recombination through in utero electroporation to produce mosaic tissue in vivo, we introduce several adaptations to procedures from other published protocols15,16,17,18,19,20,21. We provide information to improve success in breeding timed-pregnant females. We also outline our two strategies to introduce sparse and bright labeling of neurons in cortical tissue: One strategy is to titrate the levels of a single construct coding for Cre recombinase and a fluorescent marker22. Another strategy is to use the &#34;Supernova&#34; system, designed specifically with these parameters in mind23,24. Additionally, we offer improvements on producing consistent microinjection pipettes and simplifications to the in utero electroporation surgery. Finally, we outline critical steps in a simplified histological preparation that permits the analysis of dendritic spines and dendritic and axonal arbors, without further staining or immunohistochemistry.

<table>
	<tbody>
		<tr>
			<td>C57BL/6J mice</td>
			<td>The Jackson Laboratory</td>
			<td>#000664</td>
			<td>See &#34;1. Mouse set-up&#34; (step 1.1, &#34;wildtype mice&#34;)</td>
		</tr>
		<tr>
			<td>GFP.Cre empty vector</td>
			<td>AddGene</td>
			<td>#20781</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.1 &#34;single DNA construct that codes for Cre recombinase as well as a fluorescent marker&#34;). GFP.Cre empty vector was a gift from Tyler Jacks.</td>
		</tr>
		<tr>
			<td>pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (Supernova)</td>
			<td>AddGene</td>
			<td>#69138</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.1 &#34;Supernova&#34; system) and http://snsupport.webcrow.jp/. pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (Supernova) was a gift from Takuji Iwasato.</td>
		</tr>
		<tr>
			<td>pK031.TRE-Cre (Supernova)</td>
			<td>AddGene</td>
			<td>#69136</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.1 &#34;Supernova&#34; system) and http://snsupport.webcrow.jp/. pK031.TRE-Cre (Supernova) was a gift from Takuji Iwasato.</td>
		</tr>
		<tr>
			<td>pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova)</td>
			<td>AddGene</td>
			<td>#85006</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.1 &#34;Supernova&#34; system) and http://snsupport.webcrow.jp/. pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova) was a gift from Takuji Iwasato.</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit (10)</td>
			<td>Qiagen</td>
			<td>#12362</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.3 &#34;endotoxin-free plasmid purification kit&#34;)</td>
		</tr>
		<tr>
			<td>Trypan Blue powder, BioReagent grade</td>
			<td>Sigma</td>
			<td>T6146-5G</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.5 &#34;trypan blue&#34;)</td>
		</tr>
		<tr>
			<td>Sodium Chloride, ACS, 2.5 kg</td>
			<td>VWR</td>
			<td>BDH9286-2.5KG</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.5 &#34;NaCl&#34;)</td>
		</tr>
		<tr>
			<td>Potassium Chloride, ACS, 500 g</td>
			<td>VWR</td>
			<td>#97061-566</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.5 &#34;KCl&#34;)</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic, ReagentPlus, 100 g</td>
			<td>Sigma-Aldrich</td>
			<td>S0876-100G</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.5 &#34;Na2HPO4&#34;)</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic, ReagentPlus, 100 g</td>
			<td>Sigma-Aldrich</td>
			<td>P5379-100G</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.5 &#34;KH2PO4&#34;)</td>
		</tr>
		<tr>
			<td>Hydrochloric acid, ACS reagent, 500 mL</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.5 &#34;HCl&#34;)</td>
		</tr>
		<tr>
			<td>P-97 Micropipette Puller</td>
			<td>Sutter Instrument</td>
			<td>P-97</td>
			<td>See &#34;3. Pipette set-up&#34; (step 3.1 &#34;glass capillary puller&#34;)</td>
		</tr>
		<tr>
			<td>3.0 mm wide trough filament</td>
			<td>Sutter Instrument</td>
			<td>FT330B</td>
			<td>See &#34;3. Pipette set-up&#34; (step 3.1 &#34;glass capillary puller&#34;)</td>
		</tr>
		<tr>
			<td>Thin Wall Glass Capillaries, 4&#34;, 1 / 0.75 OD/ID</td>
			<td>World Precision Instruments</td>
			<td>TW100-4</td>
			<td>See &#34;3. Pipette set-up&#34; (step 3.1.1 &#34;glass capillary&#34;)</td>
		</tr>
		<tr>
			<td>Single Ply Soft-Tech Wipes, 4.5&#34;</td>
			<td>Phenix</td>
			<td>LW-8148</td>
			<td>See &#34;3. Pipette set-up&#34; (step 3.2.1 &#34;single-ply task wipe&#34;); other single-ply wipes (e.g. Kimwipes) can be used.</td>
		</tr>
		<tr>
			<td>Graefe Forceps, 7 cm, Straight, 0.7 mm 1x2 Teeth</td>
			<td>World Precision Instruments</td>
			<td>#14140</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;Graefe forceps&#34;)</td>
		</tr>
		<tr>
			<td>Iris Scissors, 11.5 cm, Straight, 12-pack</td>
			<td>World Precision Instruments</td>
			<td>#503708-12</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;iris scissors&#34;)</td>
		</tr>
		<tr>
			<td>Hartman Mosquito Forceps, 9 cm, Straight, 12-pack</td>
			<td>World Precision Instruments</td>
			<td>#503728-12</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;Hartman mosquito forceps&#34;)</td>
		</tr>
		<tr>
			<td>General Purpose Non-Woven Sponges, 2&#34; x 2&#34;, 4-ply</td>
			<td>Medrepexpress</td>
			<td>#2204-c</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;non-woven gauze sponges&#34;)</td>
		</tr>
		<tr>
			<td>Ring Tipped Forceps, 10 cm, Straight, 2.2mm ID</td>
			<td>World Precision Instruments</td>
			<td>#503203</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;ring-tipped forceps&#34;)</td>
		</tr>
		<tr>
			<td>Pyrex petri dishes complete, O.D. &#215; H 100 mm &#215; 20 mm</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3160102-12EA</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;Petri dishes&#34;)</td>
		</tr>
		<tr>
			<td>Flat Type Instrument Tray, Stainless Steel, 13-5/8&#34; x 9-3/4&#34; x 5/8&#34;</td>
			<td>Amazon</td>
			<td>B007SHGAHA</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;stainless steel tray&#34;)</td>
		</tr>
		<tr>
			<td>Platinum Tweezertrode, 5 mm</td>
			<td>BTX</td>
			<td>#45-0489</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.3 and 4.16 &#34;tweezer-type electrodes&#34;)</td>
		</tr>
		<tr>
			<td>ECM 830 Foot Pedal</td>
			<td>BTX</td>
			<td>#45-0211</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.3 and 4.17 &#34;foot pedal&#34;)</td>
		</tr>
		<tr>
			<td>ECM 830 Generator</td>
			<td>BTX</td>
			<td>#45-0052</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.3 &#34;generator&#34;)</td>
		</tr>
		<tr>
			<td>Single Animal Isoflurane Anesthesia System with Small Induction Box</td>
			<td>Harvard Apparatus</td>
			<td>#72-6468</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.4 and 4.6 &#34;nose cone&#34;, step 4.4 &#34;induction chamber&#34;)</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Hanna Pharmaceutical Supply Co</td>
			<td>#0536108691</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.7 &#34;veterinary ophthalmic ointment&#34;)</td>
		</tr>
		<tr>
			<td>Space Gel (AIMS)</td>
			<td>VWR</td>
			<td>#95059-640</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.8 &#34;sealed pouch filled with supersaturated salt solution&#34;)</td>
		</tr>
		<tr>
			<td>Hair Remover Gel Cream, Sensitive Formula</td>
			<td>Veet</td>
			<td>#062200809951</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.9 &#34;depilatory cream&#34;)</td>
		</tr>
		<tr>
			<td>10ul Low Retention Tip Starter (960 tips/pk)</td>
			<td>Phenix Research Products</td>
			<td>TSP-10LKIT</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.12 &#34;sterile 10 &#181;L micropipette tip&#34;)</td>
		</tr>
		<tr>
			<td>Aspirator tube assemblies for calibrated microcapillary pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>A5177</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.15 &#34;aspirator tube assembly&#34;)</td>
		</tr>
		<tr>
			<td>Braided Absorbable Suture, 4-0, Needle NFS-2(FS-2), 27&#34;</td>
			<td>Medrepexpress</td>
			<td>MV-J397</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.19 &#34;absorbable sutures&#34;)</td>
		</tr>
		<tr>
			<td>&#8220;LiquiVet Rapid&#8221; Tissue Adhesive</td>
			<td>Medrepexpress</td>
			<td>VG3</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.20 &#34;tissue adhesive&#34;)</td>
		</tr>
		<tr>
			<td>Hypodermic syringes, polypropylene, Luer lock tip, capacity 1.0 mL</td>
			<td>Sigma-Aldrich</td>
			<td>Z551546-100EA</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.21 &#34;1 mL syringe&#34;)</td>
		</tr>
		<tr>
			<td>BD Precisionglide syringe needles gauge 26, L 1/2 in.</td>
			<td>Sigma-Aldrich</td>
			<td>Z192392-100EA</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.21 &#34;26G, &#189;&#8221; needle&#34;)</td>
		</tr>
		<tr>
			<td>Nestlets Nesting Material</td>
			<td>Ancare</td>
			<td>NES3600</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.24 &#34;nesting materials&#34;)</td>
		</tr>
		<tr>
			<td>Sunflower Seeds, Black Oil, Sterile</td>
			<td>Bio-Serv</td>
			<td>S5137</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.24 &#34;sunflower seeds&#34;)</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 97%</td>
			<td>Alfa Aesar</td>
			<td>A11313</td>
			<td>See &#34;5. Histology&#34; (step 5.1.1 &#34;PFA&#34;)</td>
		</tr>
		<tr>
			<td>Economy Tweezers #3, 11 cm, 0.2 x 0.4 mm tips</td>
			<td>World Precision Instruments</td>
			<td>#501976</td>
			<td>See &#34;5. Histology&#34; (step 5.5 &#34;tweezers&#34;)</td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>Alfa Aesar</td>
			<td>#10752</td>
			<td>See &#34;5. Histology&#34; (step 5.8.1 &#34;agar&#34;)</td>
		</tr>
		<tr>
			<td>Single-edge razor blades, #9 blade</td>
			<td>Stanley Tools</td>
			<td>#11-515</td>
			<td>See &#34;5. Histology&#34; (step 5.9 &#34;single-edge razor blade&#34;)</td>
		</tr>
		<tr>
			<td>Specimen disc S D 50 mm</td>
			<td>Leica</td>
			<td>#14046327404</td>
			<td>See &#34;5. Histology&#34; (step 5.9 &#34;vibrating microtome specimen disc&#34;)</td>
		</tr>
		<tr>
			<td>Buffer tray S assembly</td>
			<td>Leica</td>
			<td>#1404630132</td>
			<td>See &#34;5. Histology&#34; (step 5.10 &#34;buffer tray&#34;)</td>
		</tr>
		<tr>
			<td>VT1000 S Vibratome</td>
			<td>Leica</td>
			<td>#14047235612</td>
			<td>See &#34;5. Histology&#34; (step 5.10 &#34;vibrating microtome&#34;)</td>
		</tr>
		<tr>
			<td>Double Edge Razor Blades</td>
			<td>Personna</td>
			<td>BP9020</td>
			<td>See &#34;5. Histology&#34; (step 5.10 &#34;blade&#34;)</td>
		</tr>
		<tr>
			<td>Knife Holder S</td>
			<td>Leica</td>
			<td>#14046230131</td>
			<td>See &#34;5. Histology&#34; (step 5.10 &#34;knife holder&#34;)</td>
		</tr>
		<tr>
			<td>Studio Elements Golden Taklon Short Handle Round Brush Set</td>
			<td>Amazon</td>
			<td>B0089KU6XE</td>
			<td>See &#34;5. Histology&#34; (step 5.12.1 &#34;fine tipped paintbrush&#34;)</td>
		</tr>
		<tr>
			<td>Superfrost Plus Slides</td>
			<td>Electron Microscopy Services</td>
			<td>#71869-11</td>
			<td>See &#34;5. Histology&#34; (step 5.12.1 &#34;microscope slide&#34;)</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant, 10 ml</td>
			<td>Thermofisher</td>
			<td>P36970</td>
			<td>See &#34;5. Histology&#34; (step 5.12.3-5.12.4 &#34;mountant&#34;)</td>
		</tr>
		<tr>
			<td>Cover Glass, 24 x 50 mm, No. 1</td>
			<td>Phenix Research Products</td>
			<td>MS1415-10</td>
			<td>See &#34;5. Histology&#34; (step 5.12.4 &#34;coverslip&#34;)</td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>See &#34;5. Histology&#34; (step 5.13.1 &#34;DAPI&#34;)</td>
		</tr>
		<tr>
			<td>Fixed Stage Upright Microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>See &#34;5. Histology&#34; (step 5.15 &#34;light microscope&#34;)</td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>Nikon</td>
			<td>TE2000/C2si</td>
			<td>See &#34;5. Histology&#34; (step 5.15 &#34;confocal microscope&#34;)</td>
		</tr>
		<tr>
			<td>4x objective, NA = 0.20</td>
			<td>Nikon</td>
			<td>CFI Plan Apo Lambda 4X</td>
			<td>See &#34;5. Histology&#34; (step 5.15 &#34;low-power objective&#34;)</td>
		</tr>
		<tr>
			<td>20x objective, NA = 0.75</td>
			<td>Nikon</td>
			<td>CFI Plan Apo Lambda 20X</td>
			<td>See &#34;5. Histology&#34; (step 5.15 &#34;medium-power objective&#34;)</td>
		</tr>
		<tr>
			<td>60x objective, NA = 1.40</td>
			<td>Nikon</td>
			<td>CFI Plan Apo VC 60X Oil</td>
			<td>See &#34;5. Histology&#34; (step 5.15 &#34;high-power objective&#34;)</td>
		</tr>
	</tbody>
</table>

inducing cre-lox recombination in mouse cerebral cortex through in utero electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

The single construct GFP.Cre (see list of materials) was electroporated at E15.5 and visualized at P14. Depending on the concentration of the construct and the volume of injection, a sparse or dense result can be obtained22,26. For example, injection of 1 &#181;L of 2 mg/mL GFP.Cre results in a sparse distribution of labeled cells, some of which can be bright (Figure 1A), and localized in layer II/III...

Watch this Scientific Journal Video about Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation at JoVE.com

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

The overall goal of this procedure is to generate a genetic mosaic in mouse cerebral cortex by inducing Cre-Lox recombination through in utero electroporation. This method of generating genetic mosaics can help answer key questions in neurobiology. Such as determining if a gene is necessary for a given neuronal phenotype in a cell autonomous manner.The main advantage of this technique is that the effective Cre-Lox recombination system is combined with an equally effective gene delivery technique in utero electroporation. Though this method is focused on targeting layer two-three cortical neurons, it can be used to target neurons in other cortical layers depths. When we tried to make genetic mosaics in our brain tissue, we encountered several problems in getting our experimental animals to survive after birth, so we introduced several simplifications and refinements to our technique.Prepare a pipette with a very long taper and very fine tip, typical of pipettes for embryonic stem cell injection or nuclear transfer by first inserting the glass capillary into the pipette puller. After fastening the clamps, activate the program to pulling protocol, creating a taper on the glass capillary. Ensure that the taper length is between 10 and 15 mm using a compound light microscope.Next, to break the tip of the pipette center a single-ply task wipe over a 50-mL beaker and stretch it taut with one hand. With the other hand, push the pulled pipette, taper side down, perpendicularly through the center of the wipe causing a clean break in the taper. Check the size of the opening under a compound light microscope.Then calibrate the pipette by aspirating 1 mcL of 0.4%Trypan blue and PBS into a partially filled pipette and graduate the pipette with 1-mcL markings based on the height of 1 mcL in the pipette. Use the calibrated pipette to graduate other pulled pipettes before discarding. Place the pipettes in the pipette holder free from dust and other particles.After deeply anesthetizing a pregnant mouse at E-15.5, test the righting reflex by gently tilting the induction chamber and noting the response of the mouse. If the righting reflex is absent, transfer the mouse to the surgical preparation area and administer inhaled isoflurane through a nose cone to maintain a surgical plane of anesthesia. Use forceps to test the pedal reflex to verify anesthetization.Monitor respiratory rate in effort, looking for deep, regular breathing and that mucous membranes are pink in color and moist. Apply enough veterinary ophthalmic ointment over the eyes to cover them completely for protection against drying. Massage depilatory cream onto the abdominal area with cotton swabs until the abdominal fur dissolves.Then wipe off the abdominal fur. Carefully clean off any remaining residue and depilatory cream with 70%ethanol and cotton swabs. Apply iodine-based scrub solution to the abdominal area in a circular motion.Then use a surgical drape to isolate the surgical area and maintain sterile conditions. After a making a 2-cm ventral midline incision in the skin, find the linea alba to visualize the midline of the rectus abdominis. Pull the muscle up and away from the abdomen and make a 2-cm midline incision to expose the abdominal cavity using aseptic techniques.Using a sterile 10-mcL micropipette tip, dispense carprofen into the abdominal cavity for analgesia. Next, clamp one Hartmann mosquito forceps on the left edge of the rectus abdominis incision. Rest the forceps on overturned Petri dish to the left of the incision, keeping the left side of the incision open.After clamping the right side of the incision in a similar manner, place gauze sponge around the area of the incision. Using ring forceps without an attached limit screw, pull on the uterus between any two neighboring embryos without crushing or injuring any tissue. Begin pulling out all embryos through the incision, laying them on top of the gauze sponge.Connect a calibrated pipette to the aspirator tube assembly and inject exactly 1 mcL of DNA solution prepared through the uterine wall into a lateral ventricle of each embryo. Use the thumb and forefinger to manipulate the embryos, allowing the embryos to be pushed gently against the uterine wall during injection. The lateral ventricles appear as two darker patches on the dorsal telencephalon of the embryo.Confirm successful injection by observing Trypan blue filling the lateral ventricle. After injecting all embryos, place the cathode of tweezer-type electrodes on the uterus, directly over the medial and caudal cortex to target the visual cortex. Place the anode just inferior and anterior to the embryo's head.With the foot pedal, trigger the delivery of 550-ms pulses of 50 volts separated by a 950-ms interval. After electroporating all embryos, return the uterus to the abdominal cavity in the same orientation it was found. Use saline to lubricate the uterus while guiding it manually and extremely gently, taking care not to displace embryos from their position in the uterus.Close the abdominal muscles with absorbable sutures. Use a simple interrupted stitch, tying the ends with a surgeon's knot. Close the skin layer with absorbable sutures.Apply a small amount of tissue adhesive to seal the wound. Apply tissue adhesive to the knots to prevent unfastening. Use a micropipette to remove any tissue adhesive surrounding the wound that can be aspirated.Once the tissue adhesive is dry, remove the animal from isoflurane and allow the female to recover alone in a warm cage. Continue to monitor the mouse until it is fully recovered and behaving normally. Once removed, place female back in cage with the male.Place the skull on a flat surface with single-ply task wipes moistened with 1-x PBS. Using tweezers, first remove the occipital bone. Then carefully remove the parietal bones, removing the tweezers out and away from the surface of the brain.Carefully remove any meninges to prevent damage to the cortex. Wedge the back of the tweezers under the brain along the skull to sever any cranial nerves and remove the brain. Use a single-edge razor blade to make a coronal cut approximately 0.5 mm rostral to bregma, and another coronal cut approximately 0.5 mm caudal to the tissue of interest.Tissue block is now ready for sectioning. Injection of 1 mcL of 2 mg/mL of GFP Cre results in a sparse distribution of labeled cells, some of which can be bright. Because the tissue is sectioned at 100 microns, most of the dendritic arbors are preserved.When the in utero electroporation is performed at embryonic day 15.5, labeling occurs across cortical layers two and three. Dendritic spines can be observed at high magnifications. Injection of 1.5 mcL of 2 mg/mL of GFP Cre results in very dense labeling, which can be suboptimal as it is difficult to track the source of neurites and dendritic spines.However, by selecting a bright cell in the periphery of the labeled area, it is still possible to image a neuron and its processes. Alternatively, injection of SuperNova constructs results in a sparse distribution of layer two-three neurons that are very brightly labeled. Once mastered the in utero electroporation surgery will take less than 30 minutes if it is performed properly.While attempting this procedure with other DNA constructs, it's important to remember to try different concentrations and volumes, this will help achieve the desired sparseness of labeling required for mosaic analysis. Our representative results used either a single construct or recently invented SuperNova construct. In the latter, we found that we could achieve excellent sparse labeling, while maximizing the brightness of the neurons.This technique could be adapted to create mosaic brain tissue in other ways. For example, CRISPR and Cas9 gene editing can be combined with in utero electroporation, or Cre-Lox recombination could be combined with intraventricular injection of viral vectors. After watching this video, you should have a good understanding of several simplified and refined techniques used to achieve a sparse genetic mosaic, so that you can combine Cre-Lox recombination and in utero electroporation together.We wish you the best of luck.

Research

JoVE Journal

Neuroscience

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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 ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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

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 ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...