The authors would like to thank Eva Hadzimova, Pierre Mattar and Christopher Kovach for their initial work in establishing in utero electroporation technology in the CS lab. This work was funded by a Canadian Institute of Health Research (CIHR) grant (MOP 44094) and CIHR/Foundation Fighting Blindness (FFB) Emerging Team Grant (00933-000) to CS and an Alberta Children's Hospital Research Foundation Grant to DMK. RD was supported by a CIHR Canada Hope Scholarship, RC is supported by an FFB Studentship and LML was supported by a CIHR Training Grant in Genetics and Child Development.

1. Set-up
<ol>
	<li>Set-up surgical area as shown in Fig. 1A,A'. Key components of the set-up include an Eppendorf Femtojet microinjector, Narishige micromanipulator with needle holder, Leica steromicroscope, fiber optic lighting system, ECM 830 Square Wave Electroporation System with electrodes, heating pad and a vaporizer for isoflurane anesthetic.</li> 
	<li>Clean all tools with a Bransonic Ultrasonic Cleaner using Metriclean2 Low foaming solution for sonicating surgical tools. Sterilize tools by autoclaving. Sterilized tools are then kept in Germex.</li>
	<li>Prepare syringes with warm sterile saline and sterile Lactated Ringer&#x2019;s solution.</li>
</ol>
2. Anesthesia
<ol>
	<li>Place animal inside a small (12cm x 20cm) anesthetic chamber. Using a vaporizer, deliver 5% isoflurane anesthetic and oxygen at 1L/min (Fig. 1A,A'). Notably, it is important to collect isoflurane exhaust using a scavenging system.</li> 
	<li>Remove animal from the chamber when breathing becomes deep and regular. Toe pinching and eye blink reflex tests are used to ensure full anesthesia.</li> 
	<li>Place pregnant mouse on its back on top of a heating pad (37&deg;C). The heating pad is first sterilized by wiping with 70% ethanol, and is then covered with a sterilized, lintless gauze pad. Use a respirator tube and mask apparatus to deliver isoflurane (2.25%) during surgery.</li>
	<li>Inject buprenorphine (0.1mg/kg body weight) subcutaneously under the skin at the nape of the neck.</li>
	<li>Monitor animal throughout surgery for deep and regular breathing. The animal should not gasp, breathe shallowly or rapidly. Shallow, rapid breathing may be a sign of light anaesthesia, and the flow of isoflurane should be increased. If the animal begins to gasp, reduce the flow of isoflurane. In the case of a respiratory arrest, turn isoflurane off, and increase flow of oxygen until animal recovers. Restart isoflurane at a lower concentration.</li>
</ol>
3. Animal Preparation for Surgery
<ol>
	<li>Tape forelimbs of animal to the heating pad and check again for a retraction response by pinching the hind foot with blunt forceps.</li>
	<li>Using a sterile cotton-tipped swab, spread a thick layer of depilatory cream (i.e., Nair) onto the abdomen. In firm strokes, ensure the underfur is completely coated. Leave on for an average of three minutes, checking intermittently with a sterile clean swab for hair removal. Wet a sterile gauze pad with sterile water and wipe the abdomen until all traces are gone. Repeat with chlorhexidine (i.e., Stanhexidine) skin cleaner and more water. Dry the abdomen with clean, sterile gauze.</li>
	<li>Sterilize incision site with a chlorhexidine (i.e., Stanhexidine) followed by more sterile water. Dry the abdomen with clean, sterile gauze. Repeat this step using 70% ethanol and a fresh, sterilized cotton swab.</li>
	<li>Place a sterile gauze pad over the abdomen with a slit in the middle through which the uterine horns will be pulled during surgery.</li>
</ol>
4. Incision and Exposure of Uterine Horns
<ol>
	<li>To expose the uterine horns, first locate the linea alba as the faint line under the skin at the midline. Using forceps, pull the skin away from the abdominal wall and cut a small, straight incision from the mid-abdomen downwards with skin scissors (approximately 1 cm in length).</li>
	<li>Using curved forceps and scissors, repeat the process with the peritoneum.</li>
	<li>Place sterile gauze with a small vertical slit over incision and dampen with warmed sterile saline.</li>
	<li>Slide forceps into the incision and locate the uterus. Hold open incision with forceps while removing the uterine horns.</li>
	<li>Carefully grasp the uterus between the first and second visible embryos and pull the embryos onto the gauze. Remove both sides of the uterine horns, placing onto the sterile gauze and count the number of embryos, noting any resorptions or &#x201C;unhealthy&#x201D; (e.g. smaller body size) embryos. Wet the uterine horns with saline and carefully return half of the uterus into the abdomen.</li>
</ol>
5. Injection of DNA into Ventricles
<ol>
	<li>Surgical needles are prepared with a Sutter Flaming Micropipette Puller using borosilicate micropipettes with capillaries (Program: heat=750, Pull=30, Vel= 90, Time= 150). Using a sterile scalpel blade, the needle tip is gently shaved off at a 45 degree angle. </li>
	<li>To load the needle, Pasteur pipettes are pulled to a narrow diameter under a flame. A mouth piece is attached to the loading pipette to draw up &tilde;10 &mu;l of 3mg/ml endotoxin-free DNA mixed with 0.01% methyl green (note: methyl green dye allows DNA injections to be visualized). The filled needle is then mounted on a micromanipulator stand and attached to the Femtojet injector.</li> 
	<li>Starting with the embryo closest to the ovary, circular forceps are used to gently turn the embryo within the decidua so that it is positioned &#x201C;face-forward&#x201D;, allowing injections to occur in a rostral to caudal direction. A fiber optic light source for illumination is used to help visualize the midline of the embryonic brain, which is flanked by the large lateral ventricles in rostral regions (Fig. 1B,B').</li> 
	<li>The embryo is held in position using circular forceps. The needle is positioned above the uterus so that the angle of entry into the brain is approximately 45 degrees. Using the micromanipulator, and a quick and steady motion, the needle tip is inserted through the uterine wall and into the lateral ventricle of the embryo. Injections are directed towards the lateral ventricles, 3rd ventricle or subretinal space to target the telencephalon, diencephalon and retina, respectively. Note that the embryonic brain vesicles are open at early embryonic stages such that DNA injected into the lateral ventricles will flow into the 3rd ventricle.</li>
	<li>Press the foot pedal to inject endotoxin-free DNA (1-3 &mu;L) using an Eppendorf Femtojet microinjector. A successful injection is easily visualized by spreading of the methyl green dye throughout the embryonic brain ventricles. The needle is then slowly removed by drawing back up to avoid breakage.</li>
</ol>
6. Electroporation
<ol>
	<li>The injected embryo (still within the uterus) is placed on the sterile gauze overlying the mother&#x2019;s abdomen and wet thoroughly with warmed saline. Please note** The addition of saline to the membrane surface is essential to allow efficient passage of an electrical current.</li>
	<li>GenePaddle electrodes are used to grip the uterus following the injection of DNA. Five square electric pulses of 40-50V and 50 ms are delivered at a rate of one pulse per second using an ECM8300 BTX electroporation system (Fig. 1C,C').</li> 
	<li>Cover the injected/electroporated embryo with protective wet gauze and continue to the next embryo.</li>
	<li>Upon completion of the injection/electroporation of all desired embryos, carefully push the uterine horns back into the abdomen (Fig. 1D).</li>
	<li>Carefully remove the other uterine horn and begin again with the embryo closest to the ovary.</li>
</ol>
7. Suture and Stapling
<ol>
	<li>Once all embryos are back inside the uterus, add to the abdominal cavity &tilde;2-3 ml warm saline.</li>
	<li>To close the wound, begin by removing the gauze on the abdomen and identifying the sides of the peritoneum. Begin suturing the peritoneum using black braided silk line. It is not recommended to use absorbable suture for this procedure as it is typically weaker and may lead to post-surgical bleeding and infection. Anchor the line on the peritoneum closest to the sternum and proceed to stitch away from the anchor. Ensure all sutures are tight, and that no underlying organs or skin has been inadvertently sewn into the peritoneum. Clip the remaining suture line and discard.</li>
	<li>Pull skin closed over the suture line with forceps. Using 9 mm Reflex&trade; skin closure staples, clip the skin over the wound, taking care not to staple the suture line underneath, or to pull the skin too tightly. The preferred method is to hold the skin up and away from the body with forceps, and come in at a 90 degree angle with the staple tool. Continue until wound is closed (approximately three staples). Once finished, ensure staples are tight by gently pressing together with the suture tool (Fig. 1D').</li>
</ol>
8. Post-surgical Care
<ol>
	<li>To monitor anesthetic recovery, turn female onto her stomach and turn off isoflurane. Keeping the animal&#x2019;s face inside the breathing apparatus, turn oxygen up to 3% to help recovery from anesthesia. The animal should be placed on a clean dry gauze pad on top of the heating pad to prevent hypothermia.</li>
	<li>While the animal is groggy, inject 2 mL Ringer&#x2019;s solution subcutaneously under the animal&#x2019;s neck skin.</li> 
	<li>Continue holding the animal under oxygen flow for several minutes and warming gauze pads in order to rouse the animal. Short spurts of movement from the animal should be observed as it returns to a normal breathing pattern. Observe for several minutes until animal seems conscious and awake.</li> 
	<li>Spray the female&#x2019;s abdomen with Gentamicin spray and wrap the animal in a sterile gauze pad.</li>
	<li>Transfer the animal into a sterile cage with autoclaved bedding and cardboard housing.</li>
	<li>Water bottles should be thoroughly cleaned, sterilized and filled with sterile water. To 500 mL of water add 2 mL of sulfamethazine antibiotic (25% stock).</li> 
	<li>Administer 0.05 mg/kg buprenorphine subcutaneously the morning following surgery.</li>
	<li>Monitor the female closely for the first 24 hours post-surgery. If animals are kept longer than 24 hrs, females are moved to new sterile cages on day 2.</li> 
</ol>
9. Representative Results
Neocortex 
The neocortex is the region of the CNS that is responsible for visual and sensorimotor processing and higher cognitive functioning. It is comprised of glutamatergic projection neurons and GABAergic interneurons that are derived from the dorsal and ventral telencephalon, respectively. To address the roles of different genes in neocortical development, we use in utero electroporation to either misexpress or block the expression (i.e., through the use of shRNA constructs) of different genes in the dorsal or ventral telencephalon1,3,4. To misexpress genes in the embryonic telencephalon, we use a bicistronic expression vector (pCIG2), containing a CMV-&beta;actin promoter-enhancer and an internal ribosome entry site (IRES)-enhanced green fluorescent protein (EGFP; in pCIG2) cassette, allowing transfected cells to be detected by epifluorescence (Fig. 2A-C). We used this technology to introduce expression constructs into the telencephalon from E11.5 to E15.5 (data shown is at E12.5; Fig. 2). By allowing embryos to develop several days post-electroporation, we are able to track the differentiation and migration of GFP-labeled progenitors. As differentiation proceeds, 3 days post-electroporation GFP+ cells differentiate and migrate out of the ventricular zone (vz), subventricular zone (svz) and intermediate zone (iz) and into the cortical plate (cp; Fig. 2C). If brains electroporated at E12.5 are analyzed after 24 hr, we can monitor earlier events in progenitor maturation and neuronal migration by co-labeling with markers of apical progenitors (i.e., Pax6; Fig. 2A), basal progenitors (i.e., Tbr2; Fig. 2B) and postmitotic neocortical neurons (i.e., Tbr1; Fig. 2C).
Diencephalon
The hypothalamus lies at the ventral base of the CNS and functions as a gateway between the endocrine and autonomic nervous systems. During development, the multiple nuclei that make up the mature hypothalamus differentiate from a common progenitor zone that lines the ventral-most region of the embryonic diencephalon. Currently, the molecular pathways that regulate progenitor cell proliferation, neuronal differentiation and the formation of hypothalamic nuclei are poorly understood. We used in utero electroporation to target reporter gene expression to the embryonic diencephalon by injecting DNA into the 3rd ventricle of E12 embryos.We then followed the distribution of GFP in differentiating neurons at E14. With this methodology, we have successfully targeted GFP expression to the thalamus, pre-thalamus and hypothalamus (Fig. 3A). Analysis of coronal sections reveals that we have successfully targeted GFP expression to progenitor cells that line the ventricular zone of the 3rd ventricle and neurons that migrate out to the mantle zone to form nuclei (Fig. 3B)
Retina
The retina is the neural layer of the eye, responsible for converting light photons into electrical impulses transmitted to the brain. It develops as an outpocketing of the embryonic diencephalon, and hence, is derived from the neural tube. The neural retina develops from an apical progenitor compartment that lies adjacent to the subretinal space, a cavity that separates the eye from the head mesenchyme. Targeting DNA constructs to retinal progenitors therefore requires that injections are made into a small target area. Using micromanipulators, we have managed to successfully target this small area in embryos from E13.5 to E15.5 (shown is E15.5 electroporations of pCIC with mCherry reporter; Fig. 4). If electroporated retinae are examined 24 hr post-transfection, we observe that most mCherry+ cells are located in the outer neuroblast layer (onbl), where dividing progenitors are located, and not in the retinal ganglion cell layer (gcl; inset image), where postmitotic cells localize.
<img src="/files/ftp_upload/2957/2957fig1.jpg" alt="Figure 1"/> 
Figure 1. Introduction to in utero electroporation set-up and methodology. (A) The key components of the surgical set-up include an Eppendorf Femtojet microinjector (1), Narishige micromanipulator with needle holder (2), Leica steromicroscope (3), fiber optic lighting system (4), ECM 830 Square Wave Electroporation System with electrodes (5) and heating pad (6). (A') The animal is anesthetized using a vaporizer for isoflurane anesthetic. (B,B') Briefly, after anesthesia, the uterine horns are exposed and DNA mixed with fast green is injected into the embryonic brain vesicles. A successful injection is visualized by tracking of the fast green dye (B'). (C,C') After the DNA is successfully injected, the paddle electrodes are placed on either side of the uterus, positioning the embryo so that the DNA will be pulled towards the positive pole into the brain region of choice. (D,D') Once all the desired embryos are injected, the uterus is pushed back into the abdomen (D), the peritoneum is sutured, and the skin is stapled (D'). The pregnant female is then allowed to recover and the electroporated pups are collected at the desired time points.
<img src="/files/ftp_upload/2957/2957fig2.jpg" alt="Figure 2"/> 
Figure 2. Representative example of dorsal telencephalic electroporation. (A-C) Photomicrograph of a coronal section through an E13.5 dorsal telencephalon electroporated at E12.5 with pCIG2. Electroporated GFP+ cells are analysed for their expression of markers of apical progenitors (Pax6+, red, A), basal progenitors (Tbr2+, red, B) and postmitotic neurons (Tbr1+, red, C). cp, cortical plate; svz, subventricular zone, vz; ventricular zone. 
<img src="/files/ftp_upload/2957/2957fig3.jpg" alt="Figure 3"/> 
Figure 3. Representative example of diencephalic electroporation. (A) A ventral view of an E14 brain that has been electroporated at E12 with pCIG2. GFP epifluorescence is visible in the thalamus, pre- thalamus and hypothalamus, demonstrating the spread of the microinjected pCIG2 plasmid throughout the 3rd ventricle. (B) Photomicrograph of coronal section through electroporated brain, showing the migration of GFP+ cells out of the ventricular zone and into the mantle zone, where hypothalamic nuclei will form. 3V, third ventricle; T, thalamus; Hy, hypothalamus; mz, mantle zone; Tel, telencephalon; PreT, prethalamus; vz, ventricular zone.
<img src="/files/ftp_upload/2957/2957fig4.jpg" alt="Figure 4"/> 
Figure 4. Representative example of retinal electroporation. Coronal section through an E16.5 eye electroporated with pCIC at E15.5 showing the accumulation of mCherry+ cells in the outer neuroblast layer and not in Brn3b+ retinal ganglion cells in the ganglion cell layer. The inset is a higher magnification image of the boxed area. gcl, retinal ganglion cell layer; le, lens; onbl, outer neuroblast layer; re, retina.

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No conflicts of interest declared.

In utero electroporation can be used to analyze a wide variety of developmental processes. For example, transfection of reporter genes such as GFP, mCherry or alkaline phosphatase can be used to conduct lineage tracing and neuronal migration experiments. Alternatively, Cre recombinase can be transiently expressed to selectively eliminate a floxed allele in a spatially- and/or temporally-controlled manner. Furthermore, shRNA or dominant negative constructs can be electroporated to knockdown target gene function. ...

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of reagent</th>
 <th>Company</th>
 <th>Catalogue Number</th>
 <th>Category</th>
 </tr>
 <tr>
 	<td>Fine scissors</td>
 <td>Fine Science Tools Inc.</td>
 <td>14078-10</td>
 <td>Surgical Tools</td>
 </tr>
 <tr>
 	<td>Iris scissors, curved</td>
 <td>Fine Science Tools Inc.</td>
 <td>14061-10</td>
 <td>Surgical Tools</td>
 </tr>
 <tr>
 	<td>Olsen-Hegar Ex-Delicate Needle Holder</td>
 <td>Fine Science Tools Inc.</td>
 <td>12002-12</td>
 <td>Surgical Tools</td>
 </tr>
 <tr>
 	<td>Ring forceps, 9mm</td>
 <td>Fine Science Tools Inc.</td>
 <td>11103-09</td>
 <td>Surgical Tools</td>
 </tr>
 <tr>
 	<td>Eye dressing Forcep</td>
 <td>Fine Science Tools Inc.</td>
 <td>11051-10</td>
 <td>Surgical Tools

</td>
 </tr>
 <tr>
 	<td>Dumont #7 DMX Forcep</td>
 <td>Fine Science Tools Inc.</td>
 <td>11271-30</td>
 <td>Surgical Tools

</td>
 </tr>
 <tr>
 	<td>Dumont #5 DMX Forcep</td>
 <td>Fine Science Tools Inc.</td>
 <td>11251-30</td>
 <td>Surgical Tools</td>
 </tr>
 <tr>
 	<td>Tissue forcep-Adson	</td>
 <td>Fine Science Tools Inc.</td>
 <td>11027-12</td>
 <td>Surgical Tools</td>
 </tr>
 <tr>
 	<td>Reflex Clip Applier</td>
 <td>World Precision Instrument</td>
 <td>500343</td>
 <td>Surgical Tools</td>
 </tr>
 <tr>
 	<td>Perforated Spoon, 15 mm diameter</td>
 <td>Fine Science Tools Inc.</td>
 <td>10370-18</td>
 <td>Surgical Tools</td>
 </tr>
 <tr>
 	<td>Autoclip Remover</td>
 <td>Mikron</td>
 <td>427637</td>
 <td>Surgical Tools</td>
 </tr>
 <tr>
 	<td>Silk Black Braided Suture</td>
 <td>Ethicon Inc. </td>
 <td>K871</td>
 <td>Surgical Tools

</td>
 </tr>
 <tr>
 	<td>Reflex Skin Closure Stainless Steel Wound Clips</td>
 <td>World Precision Instruments</td>
 <td>500346</td>
 <td>Surgical Tools</td>
 </tr>
 <tr>
 	<td>ECM 830 Square Wave Electroporation System</td>
 <td>VWR-CanLab</td>
 <td>58018-004</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Tweezers w/Variable Gap 2 Round 5mm Platinum Plate Electrode</td>
 <td>Protech International Inc.</td>
 <td>CUY650P5

</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Tweezers w/Variable Gap 2 Round 7mm Platinum Plate Electrode

</td>
 <td>Protech International Inc.</td>
 <td>CUY650P7

</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Eppendorf Femtojet Microinjector</td>
 <td>VWR CanLab</td>
 <td>CA62111-488</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Foot Control for Eppendorf Femtojet Microinjector</td>
 <td>VWR CanLab</td>
 <td>CAACCESS (misc.)</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Bransonic Ultrasonic Cleaner 
Model 1510R-DTH</td>
 <td>VWR CanLab	</td>
 <td>CA33995-534
CPN-952-118

</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Sutter P97 Micropipet Puller </td>
 <td>Sutter Instrument,
 Carsen Group Inc.
</td>
 <td>P-97</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Micropipettes &#x2013; Borosilicate with filament 
O.D.: 1mm, I.D.: 0.78 mm, 10 cm length
</td>
 <td>Sutter Instrument</td>
 <td>BF100-78-10</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>3-Axis Coarse Manipulator</td>
 <td>Carl Zeiss Canada Inc.</td>
 <td>M-152

</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Magnetic Holding Device for micromanipulator</td>
 <td>World Precision Instruments</td>
 <td>M1</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Steel Base Plate for micromanipulator</td>
 <td>World Precision Instruments</td>
 <td>5052</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Micropipette Holder </td>
 <td>World Precision Instruments</td>
 <td>MPH3</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Micropipette Handle</td>
 <td>World Precision Instruments</td>
 <td>5444

</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Stereomicroscope</td>
 <td>Leica</td>
 <td>MZ6</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Vaporizer for isoflurane anesthetic

</td>
 <td>Porter Instruments Company

</td>
 <td>MODEL 100-F

</td>
 <td>Instruments</td>
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 	<td>Metriclean2 Low foaming solution for sonicating surgical tools</td>
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 <td>Distributed by Church &amp; Dwight Co., Inc.</td>
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 	<td>Stanhexidine 4% w/v skin cleaner</td>
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 	<td>Buprenorphine (Temgesic) analgesic</td>
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 <td>Baxter Corporation</td>
 <td>DIN# 0061085</td>
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 <td>DIN# 01924303</td>
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 	<td>Inhalation Anesthetic &#x2013; Isoflurane USP</td>
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Partners of Canada Inc.</td>
 <td>DIN# 02237518

</td>
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 	<td>Fast Green FCF</td>
 <td>Sigma-Aldrich</td>
 <td>F7252

</td>
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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.

Watch this Scientific Journal Video about Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation at JoVE.com

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.

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 ...

efficient-gene-delivery-into-multiple-cns-territories-using-utero

Research

JoVE Journal

Neuroscience

19.8K Views.  University of Calgary. 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.

Video: Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

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 ...

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 ...

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

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

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

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

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

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 mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

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

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

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 has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

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

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

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 ...