Name: inducing cre-lox recombination in mouse cerebral cortex through in utero electroporation
Uploaded: 2017-11-17T15:00:00.000+00:00
Duration: 10 min 29 s
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.).

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The authors have no conflicts of interest to disclose.

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 electroporation15,16,17,18,19,20,21,34. In our approach, targeting the cerebral cortex is consistent and reproducible because we use 5 mm diameter electroporation electrodes that can be placed over a large area of the developing telencephalon. To target alternative sites in the brain (e.g. diencephalon, retina), or for more specific targeting of regions within the cerebral cortex, procedures and electrodes designed explicitly for this purpose may be used15,16. We also provide a single construct that provides bright labeling and guarantees that every fluorescently-labeled neuron contains Cre recombinase. A disadvantage of using a single construct to achieve bright labeling is that the labeled population can be too dense (Figure 2C), but this can be resolved by imaging cells at the periphery of the labeled area (Figure 2D). Alternatively, expression of a fluorescent construct can be designed to depend on Cre recombinase expression17, but low expression levels may require immunostaining for the fluorescent marker. This limitation is addressed by the &#34;Supernova&#34; system of Cre-dependent fluorescent marker expression (Figure 3A)23,24.
In addition to combining Cre-lox recombination with in utero electroporation, we offer several improvements for optimal breeding of timed-pregnant females. It is important to keep the mice in a holding room that is free from noises and vibrations caused by equipment such as laminar flow hoods. Enrich the environment with plastic igloos, nesting material, and dietary supplements like sunflower seeds (see list of materials). Apart from environmental considerations, we have noticed that the first litter of a female typically has the lowest survival rate. Thus, our protocol suggests waiting until the second pregnancy of the female to perform in utero electroporation. While this strategy increases the survival of the litter, it also tends to increase litter size, which can lengthen surgical time and thus lower the success of the surgery. Therefore, if encountering a particularly large number of embryos (e.g. more than 8), we suggest skipping the electroporation on some of the embryos, especially embryos closest to the ovaries and cervix, where the tissue is most prone to injury. Furthermore, using the same environmental enrichment strategy mentioned above (plastic igloos, nesting material, dietary supplements) tends to increase the survival of litters after birth. It is also important to note that estrus can occur in the female a few hours after parturition of the first litter. In this case, allow the female to give birth to her second litter, then separate after weaning in order to attempt timed mating. When looking for vaginal plugs, it is good practice to separate the female even if a plug is not found, particularly if the female was in estrus the night before. Plugs in certain strains are difficult to spot, and conception may have already occurred.
Another area of improvement is in producing adequate microinjection pipettes for in utero electroporation. Pulling pipettes for DNA injection into lateral ventricles is a critical step. Here, we provide a detailed protocol for setting up pipettes with a consistent tip size and rough tip edge. Other methods include breaking back the tip with a scalpel blade15 or pinching off the tip with forceps16,17. Note that if pipette tips are too small, they will correctly puncture the uterine wall but injection flow rates will be too low, lengthening the time in which the pipette is lodged in the embryonic brain. If pipette tips are too large, they may damage the uterine wall and embryonic brain. If pipette tips are too smooth, they will cause a large divot in the uterine wall and/or embryonic skull before breaking through, possibly damaging the embryo. Practice creating a rough break to produce a 20-25 &#181;m tip and inspect tips under a simple light microscope (e.g. with a 10X objective) until a consistent result is achieved. Testing that liquid can be pipetted at a reasonable rate (e.g. ~0.5 &#181;L/s) with an aspirator tube assembly is another method of ensuring that tip size is within an acceptable range. Because the injection pipettes are calibrated and graduated, the exact amount of DNA delivered to the lateral ventricle is known. This is the most critical step for achieving a desired sparseness reproducibly to an order of magnitude. In other words, a given concentration should yield labeling within a particular range, e.g. 10 to 100 labeled neurons.
The most important step in the protocol is the in utero electroporation procedure. Be extremely gentle while exposing the uterus. When pulling out the last embryos from the abdominal cavity, make sure not to pull on the ovaries or cervix. Using the thumb and forefinger during microinjection and electroporation allows gentle, dexterous manipulation of the embryos to reveal the lateral ventricle and allows the embryos to be pushed gently against the uterine wall. An extremely gentle touch is critical when handling the uterus and embryos. If it is difficult to achieve a gentle touch, use ring forceps with a built-in limit screw to prevent the forceps from clamping down on an embryo or the uterus, causing tissue damage. A further consideration is to administer both the carprofen and buprenorphine immediately before the surgery, a practice that appears to provide effective pain management during in utero surgery without affecting embryonic survival rates35.
In following our simplified histological procedure, several steps are critical. After perfusing experimental brains with fixative, keep the brain intact in the skull to protect the brain tissue from damage during post-fixation. While it is possible to store the brain in 1% PFA for days to weeks, note that fluorescence will decrease as the PFA solution polymerizes. Slicing 100 &#181;m coronal sections is typically thin enough to permit confocal microscopy through the entire section while preserving much of the dendritic and axonal morphology. If fixation occurred properly, brains from animals older than P13 can be mounted onto the vibrating microtome simply with cyanoacrylate glue. However, if the brain is too pliable while being cut by the vibrating microtome, glue down a small agar or agarose block behind the brain to prevent it from bending while it is being sectioned. If the problem is not resolved, embed the brain in agar to form a block to cut with the vibrating microtome, as suggested for younger postnatal brains (step 5.8).
This method combines the Cre-lox recombination system with in utero electroporation in order to generate mosaics that can be used to study the cell-autonomous function of genes in neurons. This method could also be configured to support in utero electroporation of gene editing constructs (e.g. CRISPR/Cas9)6, or other site-specific recombinases (e.g. Flp/FRT or Dre/rox24), which could all be designed to produce mosaic brain tissue. One promising alternative technique that could be used to produce mosaic tissue is viral delivery via intraventricular injection to neonatal mice36. Combined with intraventricular injection at embryonic ages as demonstrated here and elsewhere15,16,17,18,19,20,21,22,37, viral vectors coding for Cre recombinase could be delivered before birth to infect floxed progenitor cells at the ventricular zone. One critical consideration would be to ensure that the viral vectors are diluted sufficiently to achieve sparse excision and labeling. However, this would also result in lower copy numbers of the construct being delivered, including fluorescent markers essential for neuroanatomical observations (e.g. dendritic spines or arborization)24. To counter this pitfall, new constructs using the same strategy as the &#34;Supernova&#34; constructs demonstrated here could be used to achieve sparse labeling without a corresponding decrease in the brightness of labeled cells24. Another critical consideration with viral delivery is to ensure that delivered constructs have promoters specific to the cell population being studied (e.g. excitatory pyramidal neurons). Intraventricular injection of viral vectors causes infection of all tissue surrounding the ventricles, including not only the ventricular zone of the dorsal telencephalon where cortical and hippocampal excitatory neurons are generated, but also the medial and lateral ganglionic eminences where many cortical interneurons are born34. Another feature of in utero electroporation compared to viral delivery of constructs is its relatively narrow time window of delivery. Viruses injected into the lateral ventricles can persist after the surgery, continuing to infect cells lining the ventricular zone. In contrast, electroporation occurs in seconds, targeting a far more specific set of cells. This may be an advantage or disadvantage to the investigator, depending on the subpopulation of cells to be studied.
Our approach supports Cre-lox recombination by introduction of either a single construct or &#34;Supernova&#34; constructs. In the case of the &#34;Supernova&#34; constructs, we urge investigators to become familiar with the advantages and limitations of using this strategy. For example, the timing of Cre deletion has been demonstrated to occur within 2 days in layer II/III excitatory neurons of the cerebral cortex24. Thus, it is plausible that Cre excision could occur over the 48 h following electroporation, rather than immediately following electroporation. Therefore, other forms of corroborating timing and location of Cre excision (e.g. using a Cre reporter mouse line) should be used to complement the use of this new technique, especially in studies where Cre excision within a small timeframe is a critical consideration. Another potential pitfall is that a small percentage of unlabeled cells in a &#34;Supernova&#34; experiment could express Cre recombinase. For example, in Figure 3, we co-electroporated two plasmids: a high concentration of CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE, and a low concentration of TRE-Cre. Because TRE is a leaky promoter, Cre recombinase could be expressed in cells that received the TRE-Cre plasmid but not the loxP-stop-loxP-EGFP-tTA plasmid, though this would be a rare occurrence. In an experiment where it is essential that all unlabeled cells have no Cre-mediated excision whatsoever, a &#34;Supernova&#34; experiment may need to be supplemented with a control experiment in which Cre recombinase expression outside of labeled neurons is assessed through other means (e.g. Cre recombinase immunohistochemistry).
In summary, our protocol is easily modified to accommodate these new constructs, making in utero electroporation an even more useful and adaptable method for mosaic analysis. Thus, in utero electroporation can be combined with the power of genetic recombination in multiple ways to study mosaic brain tissue in vivo.

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 &quot;1. Mouse set-up&quot; (step 1.1, &quot;wildtype mice&quot;)</td></tr><tr><td>GFP.Cre empty vector</td><td>AddGene</td><td>#20781</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;single DNA construct that codes for Cre recombinase as well as a fluorescent marker&quot;). 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 &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; 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 &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; 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 &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; 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 &quot;2. DNA set-up&quot; (step 2.3 &quot;endotoxin-free plasmid purification kit&quot;)</td></tr><tr><td>Trypan Blue powder, BioReagent grade</td><td>Sigma</td><td>T6146-5G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;trypan blue&quot;)</td></tr><tr><td>Sodium Chloride, ACS, 2.5 kg</td><td>VWR</td><td>BDH9286-2.5KG</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;NaCl&quot;)</td></tr><tr><td>Potassium Chloride, ACS, 500 g</td><td>VWR</td><td>#97061-566</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;KCl&quot;)</td></tr><tr><td>Sodium phosphate dibasic, ReagentPlus, 100 g</td><td>Sigma-Aldrich</td><td>S0876-100G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&quot;)</td></tr><tr><td>Potassium phosphate monobasic, ReagentPlus, 100 g</td><td>Sigma-Aldrich</td><td>P5379-100G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&quot;)</td></tr><tr><td>Hydrochloric acid, ACS reagent, 500 mL</td><td>Fisher Scientific</td><td>A144-500</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;HCl&quot;)</td></tr><tr><td>P-97 Micropipette Puller</td><td>Sutter Instrument</td><td>P-97</td><td>See &quot;3. Pipette set-up&quot; (step 3.1 &quot;glass capillary puller&quot;)</td></tr><tr><td>3.0 mm wide trough filament</td><td>Sutter Instrument</td><td>FT330B</td><td>See &quot;3. Pipette set-up&quot; (step 3.1 &quot;glass capillary puller&quot;)</td></tr><tr><td>Thin Wall Glass Capillaries, 4&quot;, 1 / 0.75 OD/ID</td><td>World Precision Instruments</td><td>TW100-4</td><td>See &quot;3. Pipette set-up&quot; (step 3.1.1 &quot;glass capillary&quot;)</td></tr><tr><td>Single Ply Soft-Tech Wipes, 4.5&quot;</td><td>Phenix</td><td>LW-8148</td><td>See &quot;3. Pipette set-up&quot; (step 3.2.1 &quot;single-ply task wipe&quot;); 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 &quot;4. In utero electroporation&quot; (step 4.1 &quot;Graefe forceps&quot;)</td></tr><tr><td>Iris Scissors, 11.5 cm, Straight, 12-pack</td><td>World Precision Instruments</td><td>#503708-12</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;iris scissors&quot;)</td></tr><tr><td>Hartman Mosquito Forceps, 9 cm, Straight, 12-pack</td><td>World Precision Instruments</td><td>#503728-12</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Hartman mosquito forceps&quot;)</td></tr><tr><td>General Purpose Non-Woven Sponges, 2&quot; x 2&quot;, 4-ply</td><td>Medrepexpress</td><td>#2204-c</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;non-woven gauze sponges&quot;)</td></tr><tr><td>Ring Tipped Forceps, 10 cm, Straight, 2.2mm ID</td><td>World Precision Instruments</td><td>#503203</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;ring-tipped forceps&quot;)</td></tr><tr><td>Pyrex petri dishes complete, O.D. &amp;times; H 100 mm &amp;times; 20 mm</td><td>Sigma-Aldrich</td><td>CLS3160102-12EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Petri dishes&quot;)</td></tr><tr><td>Flat Type Instrument Tray, Stainless Steel, 13-5/8&quot; x 9-3/4&quot; x 5/8&quot;</td><td>Amazon</td><td>B007SHGAHA</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;stainless steel tray&quot;)</td></tr><tr><td>Platinum Tweezertrode, 5 mm</td><td>BTX</td><td>#45-0489</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 and 4.16 &quot;tweezer-type electrodes&quot;)</td></tr><tr><td>ECM 830 Foot Pedal</td><td>BTX</td><td>#45-0211</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 and 4.17 &quot;foot pedal&quot;)</td></tr><tr><td>ECM 830 Generator</td><td>BTX</td><td>#45-0052</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 &quot;generator&quot;)</td></tr><tr><td>Single Animal Isoflurane Anesthesia System with Small Induction Box</td><td>Harvard Apparatus</td><td>#72-6468</td><td>See &quot;4. In utero electroporation&quot; (step 4.4 and 4.6 &quot;nose cone&quot;, step 4.4 &quot;induction chamber&quot;)</td></tr><tr><td>Ophthalmic ointment</td><td>Hanna Pharmaceutical Supply Co</td><td>#0536108691</td><td>See &quot;4. In utero electroporation&quot; (step 4.7 &quot;veterinary ophthalmic ointment&quot;)</td></tr><tr><td>Space Gel (AIMS)</td><td>VWR</td><td>#95059-640</td><td>See &quot;4. In utero electroporation&quot; (step 4.8 &quot;sealed pouch filled with supersaturated salt solution&quot;)</td></tr><tr><td>Hair Remover Gel Cream, Sensitive Formula</td><td>Veet</td><td>#062200809951</td><td>See &quot;4. In utero electroporation&quot; (step 4.9 &quot;depilatory cream&quot;)</td></tr><tr><td>10ul Low Retention Tip Starter (960 tips/pk)</td><td>Phenix Research Products</td><td>TSP-10LKIT</td><td>See &quot;4. In utero electroporation&quot; (step 4.12 &quot;sterile 10 &amp;micro;L micropipette tip&quot;)</td></tr><tr><td>Aspirator tube assemblies for calibrated microcapillary pipettes</td><td>Sigma-Aldrich</td><td>A5177</td><td>See &quot;4. In utero electroporation&quot; (step 4.15 &quot;aspirator tube assembly&quot;)</td></tr><tr><td>Braided Absorbable Suture, 4-0, Needle NFS-2(FS-2), 27&quot;</td><td>Medrepexpress</td><td>MV-J397</td><td>See &quot;4. In utero electroporation&quot; (step 4.19 &quot;absorbable sutures&quot;)</td></tr><tr><td>&amp;ldquo;LiquiVet Rapid&amp;rdquo; Tissue Adhesive</td><td>Medrepexpress</td><td>VG3</td><td>See &quot;4. In utero electroporation&quot; (step 4.20 &quot;tissue adhesive&quot;)</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 &quot;4. In utero electroporation&quot; (step 4.21 &quot;1 mL syringe&quot;)</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 &quot;4. In utero electroporation&quot; (step 4.21 &quot;26G, &amp;frac12;&amp;rdquo; needle&quot;)</td></tr><tr><td>Nestlets Nesting Material</td><td>Ancare</td><td>NES3600</td><td>See &quot;4. In utero electroporation&quot; (step 4.24 &quot;nesting materials&quot;)</td></tr><tr><td>Sunflower Seeds, Black Oil, Sterile</td><td>Bio-Serv</td><td>S5137</td><td>See &quot;4. In utero electroporation&quot; (step 4.24 &quot;sunflower seeds&quot;)</td></tr><tr><td>Paraformaldehyde, 97%</td><td>Alfa Aesar</td><td>A11313</td><td>See &quot;5. Histology&quot; (step 5.1.1 &quot;PFA&quot;)</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 &quot;5. Histology&quot; (step 5.5 &quot;tweezers&quot;)</td></tr><tr><td>Agar powder</td><td>Alfa Aesar</td><td>#10752</td><td>See &quot;5. Histology&quot; (step 5.8.1 &quot;agar&quot;)</td></tr><tr><td>Single-edge razor blades, #9 blade</td><td>Stanley Tools</td><td>#11-515</td><td>See &quot;5. Histology&quot; (step 5.9 &quot;single-edge razor blade&quot;)</td></tr><tr><td>Specimen disc S D 50 mm</td><td>Leica</td><td>#14046327404</td><td>See &quot;5. Histology&quot; (step 5.9 &quot;vibrating microtome specimen disc&quot;)</td></tr><tr><td>Buffer tray S assembly</td><td>Leica</td><td>#1404630132</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;buffer tray&quot;)</td></tr><tr><td>VT1000 S Vibratome</td><td>Leica</td><td>#14047235612</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;vibrating microtome&quot;)</td></tr><tr><td>Double Edge Razor Blades</td><td>Personna</td><td>BP9020</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;blade&quot;)</td></tr><tr><td>Knife Holder S</td><td>Leica</td><td>#14046230131</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;knife holder&quot;)</td></tr><tr><td>Studio Elements Golden Taklon Short Handle Round Brush Set</td><td>Amazon</td><td>B0089KU6XE</td><td>See &quot;5. Histology&quot; (step 5.12.1 &quot;fine tipped paintbrush&quot;)</td></tr><tr><td>Superfrost Plus Slides</td><td>Electron Microscopy Services</td><td>#71869-11</td><td>See &quot;5. Histology&quot; (step 5.12.1 &quot;microscope slide&quot;)</td></tr><tr><td>ProLong Diamond Antifade Mountant, 10 ml</td><td>Thermofisher</td><td>P36970</td><td>See &quot;5. Histology&quot; (step 5.12.3-5.12.4 &quot;mountant&quot;)</td></tr><tr><td>Cover Glass, 24 x 50 mm, No. 1</td><td>Phenix Research Products</td><td>MS1415-10</td><td>See &quot;5. Histology&quot; (step 5.12.4 &quot;coverslip&quot;)</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td><td>Sigma-Aldrich</td><td>D9542</td><td>See &quot;5. Histology&quot; (step 5.13.1 &quot;DAPI&quot;)</td></tr><tr><td>Fixed Stage Upright Microscope</td><td>Olympus</td><td>BX51WI</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;light microscope&quot;)</td></tr><tr><td>Laser Scanning Confocal Microscope</td><td>Nikon</td><td>TE2000/C2si</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;confocal microscope&quot;)</td></tr><tr><td>4x objective, NA = 0.20</td><td>Nikon</td><td>CFI Plan Apo Lambda 4X</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;low-power objective&quot;)</td></tr><tr><td>20x objective, NA = 0.75</td><td>Nikon</td><td>CFI Plan Apo Lambda 20X</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;medium-power objective&quot;)</td></tr><tr><td>60x objective, NA = 1.40</td><td>Nikon</td><td>CFI Plan Apo VC 60X Oil</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;high-power objective&quot;)</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 (Figure 1B). Because the tissue is 100 &#181;m thick, most of the dendritic arbors are preserved (Figure 1C). Dendritic spines can be observed at high magnifications (60X; Figure 1D). Injection of 1.5 &#181;L at the same concentration results in very dense labeling (Figure 2A) in layer II/III (Figure 2B), which can be sub-optimal as it is difficult to track the source of neurites and dendritic spines (Figure 2C). However, it is still possible to image a neuron (Figure 2D) and its processes (Figure 2E) by selecting a bright cell in the periphery of the labeled area (Figure 2D).
Finally, it is possible to maximize the brightness of neurons while maintaining a sparse distribution of labeled cells. Here, Supernova-GFP (see list of materials) was electroporated at E15.5 and visualized at P23. Note that, based on observations of brain tissue taken at various postnatal ages, there does not appear to be an effect of age on the brightness of any fluorescent constructs used here23,24,26. Injection of 1 &#181;L of a mixture containing 1 mg/mL Sn-GFP (CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE) and 10 &#181;g/mL TRE-Cre results in a sparse distribution of mostly bright cells (Figure 3A) in layer II/III (Figure 3B). Dendritic and axonal processes (Figure 3B) and dendritic spines (Figure 3C) can be visualized. In our experience, targeting with either a single construct such as GFP.Cre or with &#34;Supernova&#34; constructs will reproducibly yield expression in at least 75% of electroporated embryos.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56675/56675fig1.jpg" /> 
Figure 1: Sparse and bright expression after in utero electroporation with a single construct containing GFP and Cre recombinase. (A) Low-magnification (4X) fluorescence micrograph of cerebral cortex at P23, after electroporation at E15.5. (B) Medium-magnification (20X) fluorescence micrograph of DAPI nuclear stain, revealing cortical lamination (layers I, II/III, IV, and layers deep to IV). (C) Medium-magnification (20X) fluorescence micrograph of a single neuron. (D) High-magnification (60X) fluorescence micrograph of dendritic spines. Scale bars =&#160;200 &#181;m (A); 100 &#181;m (B,C); and 5 &#181;m (D). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56675/56675fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56675/56675fig2.jpg" /> 
Figure 2: Dense and bright expression after in utero electroporation with a single construct containing GFP and Cre recombinase. (A) Low-magnification (4X) fluorescence micrograph of cerebral cortex at P14, after electroporation at E15.5. (B) Medium-magnification (20X) fluorescence micrograph of DAPI nuclear stain, revealing cortical lamination (layers I, II/III, IV, and layers deep to IV). (C) Medium-magnification (20X) fluorescence micrograph of neurons in the area of densest labeling. (D) Medium-magnification (20X) fluorescence micrograph of neurons taken at the periphery of the area of densest labeling. (E) High-magnification (60X) fluorescence micrograph of dendritic spines. Scale bars =&#160;200 &#181;m (A); 100 &#181;m (B, C, D); and 5 &#181;m (E). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56675/56675fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56675/56675fig3.jpg" /> 
Figure 3: Sparse and bright expression after in utero electroporation with Supernova-GFP constructs containing GFP and Cre recombinase. (A) Low-magnification (4X) fluorescence micrograph of cerebral cortex at P14, after electroporation at E15.5. (B) Medium-magnification (20X) fluorescence micrograph of DAPI nuclear stain, revealing cortical lamination (layers I, II/III, IV, and layers deep to IV). (C) Medium-magnification (20X) fluorescence micrograph of a single neuron. (D) High-magnification (60X) fluorescence micrograph of dendritic spines. Scale bars =&#160;200 &#181;m (A); 100 &#181;m (B,C); and 5 &#181;m (D). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56675/56675fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

11.6K Views.  James Madison University. 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.

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

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 ...

Developmental Biology

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Biology

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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 molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

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

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 first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

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

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

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

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 this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

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 ...

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 system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

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

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

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

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

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

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

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

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

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

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

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

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

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes