Name: cortex-, hippocampus-, thalamus-, hypothalamus-, lateral septal nucleus- and striatum-specific in utero electroporation in the c57bl/6 mouse
Uploaded: 2016-01-18T14:00:00.000+00:00
Duration: 6 min
Description: Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

Technical supported by Melanie Pfeifer and Nikolai Schmarowski (Institute for Microscopic Anatomy and Neurobiology, University Medical Center Mainz).

Ethics Statement: The handling of the mice and the experimental procedures were conducted in accordance with European, national and institutional guidelines for animal care.
1. In Utero Electroporation
Note: In utero electroporation was performed as previously published12,14. Therefore, the method is only described briefly in the following (Figure 1).
<ol>
	<li>Preparations
	<ol>
		<li>Prepare Fast Green colored endotoxin-free advanced transfection-grade plasmid solution (containing 1,5 pCAGGS) as previously described12.</li>
		<li>Pull and grind (35&#176;angle) borosilicate glass capillaries (0.8-0.9 diameter) for injections.</li>
		<li>Sterilize instruments.</li>
	</ol>
	</li>
	<li>Surgery
	<ol>
		<li>Administer analgesics (carprofen, 4 mg/kg bodyweight, s.c., 24 hr depot) at least 30 min before starting the surgery.</li>
		<li>Keep the length of the anesthesia to a maximum of 35-45 min (Stage III - stage of surgical anesthesia according to Guedel15 - maximal 25-30 min).</li>
		<li>Anesthetize timed pregnant mice with isoflurane (induction in a chamber: 2.8%, surgery via mask: 2.5%). Confirm anesthesia by unresponsiveness to toe-pinch.</li>
		<li>Apply eye ointment to prevent eye-dryness during anesthesia.</li>
		<li>Sterilize the surgical area with 7.5% Providone-Iodine solution and cover the mouse with sterile gauze (moist with 0.9% benzyl alcohol saline solution) with only the surgical area exposed.</li>
		<li>Cut open the abdominal cavity with a scalpel (skin incision: 1.5-2 cm, muscle incision: 1-1.5 cm).</li>
		<li>Preserve the opened abdominal cavity from drying out by moistening with warmed 0.9% benzyl alcohol saline solution or other sterile isotonic solution as directed by veterinary staff or committee.&#160;</li>
		<li>Carefully extract uterine horns (using ring forceps, do not touch the uterus with fingers).</li>
	</ol>
	</li>
	<li>Injection of DNA and electroporation. Note: The exact details for targeting specific regions are indicated in section 2.
	<ol>
		<li>Slowly inject the colored DNA solution into the ventricle as indicated in Figure 1 and section 2 via the prepared borosilicate capillaries.</li>
		<li>Apply the appropriate voltage for the corresponding embryonic stage (e.g., 36-38 V for E14) via specialized forceps-type platinum electrodes (interval cycle length 50 msec, interval pause 950 msec).&#160;Pulse the electroporation paddles during the application of the voltage to minimize damage of the uterine wall.</li>
	</ol>
	</li>
	<li>Post electroporation/ post-operative care
	<ol>
		<li>Carefully replace the uterine horns in the abdominal cavity.</li>
		<li>Suture the muscle and the skin incision separately.</li>
		<li>Let the mouse rest on a thermal support device&#160;for recovery.</li>
		<li>Supervise the mouse during recovery (behavior, feed and water consumption), 4 hr, 24 hr and 48 hr after surgery. If necessary, apply additional analgesics (carprofen, 4 mg/kg bodyweight, 24 hr depot) for further 2 days.</li>
	</ol>
	</li>
</ol>
2. Transfection of Specific Cerebral Areas
Note: The position of the positive pole is shown as the angle between the vertical centerline through the ventricle filled with the DNA solution (the right or third ventricle) and the position of the positive pole. When filling the left ventricle the positions are mirror-inverted. In Figure 4 the positions of the ear primordial are shown, which provide important reference points.
<ol>
	<li>Transfection of cortical areas (Figure 2A, Table 1).
	<ol>
		<li>Perform surgery as described in section 1.2. For the in utero electroporation of cortical areas use E14 embryos.</li>
		<li>Inject 1.5-2 &#181;l DNA-solution containing 4 &#181;g DNA in the right lateral ventricle via the prepared borosilicate capillaries.
		<ol>
			<li>Carefully position the embryo by using ring forceps so that there are no visible vessels above the puncture site.</li>
			<li>Slowly inject the DNA-solution approximately 0.75 mm coronal from the lambdoidal suture and 0.5 mm lateral from the sagittal suture (Figure 3). Ensure that the insertion depth is 1.2 mm (E14, measured from the uterine wall). Check for a blue-green colouring with a sharp demarcation.</li>
		</ol>
		</li>
		<li>Apply voltage via the specialized forceps-type platinum electrodes (interval cycle length 50 msec, interval pause 950 msec).
		<ol>
			<li>Use a 3 or 5 mm electrode paddle.</li>
			<li>Use a voltage ranging from 36-38 V.</li>
			<li>Place the center of the electrodes just anterior of the ear primordia.</li>
			<li>To transfect the retrosplenial cortex use angles from 330-0&#176;, to transfect the motor cortex 0-40&#176;, to transfect the somatosensory cortex 40-95&#176;, to transfect the piriform cortex 95-110&#176; (Figure 2A).</li>
		</ol>
		</li>
		<li>Perform post electroporation/ post-operative care as described in section 1.4. Sacrifice the mice and analyze the embryos 4 days after electroporation (E18) as described in section 3.</li>
	</ol>
	</li>
	<li>Transfection of the hippocampus (Figure 2B, Table 1).
	<ol>
		<li>Perform surgery as described in section 1.2. For the in utero electroporation of hippocampal formation use E15 embryos.</li>
		<li>Inject 2 &#181;l DNA-solution containing 4 &#181;g DNA in the right lateral ventricle via the prepared borosilicate capillaries.</li>
		<li>Apply voltage via the specialized forceps-type platinum electrodes (interval cycle length 50 msec, interval pause 950 msec).
		<ol>
			<li>Use a 3 or 5 mm electrode paddle.</li>
			<li>Use a voltage ranging from 38-40.</li>
			<li>Place the center of the negative pole just anterior of the ear primordium and the center of the positive pole at the middle of the ear primordium.</li>
			<li>To transfect the dentate gyrus use angles from 190-210&#176;, to transfect the cornu ammonis (CA)3 210-230&#176;, to transfect the CA2 230-250&#176;, to transfect the CA1 250-275&#176; (Figure 2B).</li>
		</ol>
		</li>
		<li>Perform post electroporation/ post-operative care as described in section 1.4. Perform analysis after complete hippocampal formation (after birth, p21) as described in section 3.</li>
	</ol>
	</li>
	<li>Transfection of the lateral septal nucleus and striatum (Figure 2C, Table 1) 
	Note: The angles overlap with those for transfecting the hippocampus. To effectively transfect the lateral septal nucleus and the striatum without unwanted hippocampus transfection earlier embryonic stages (E12) have to be used.
	<ol>
		<li>Perform surgery as described in section 1.2. For the in utero electroporation of the lateral septal nucleus and striatum use E12 embryos.</li>
		<li>Inject 1 &#181;l DNA-solution containing 4 &#181;g DNA in the right lateral ventricle via the prepared borosilicate capillaries.</li>
		<li>Apply voltage via the specialized forceps-type platinum electrodes (interval cycle length 50 msec, interval pause 950 msec).
		<ol>
			<li>Use a 0.5 mm electrode.</li>
			<li>Use 33 V.</li>
			<li>Place the center of the electrodes at the level of the ear primordia.</li>
			<li>To transfect the lateral septal nucleus use angles from 100-170&#176;, to transfect the striatum 170-240&#176; (Figure 2C).</li>
		</ol>
		</li>
		<li>Perform post electroporation/ post-operative care as described in section 1.4. Sacrifice the mice and analyze the embryos 6 days after electroporation (E18) as described in section 3.</li>
	</ol>
	</li>
	<li>Transfection of the thalamus and hypothalamus (Figure 2D, Table 1)
	<ol>
		<li>Perform surgery as described in section 1.2. For the in utero electroporation of the thalamus and hypothalamus use E12-E13 embryos.</li>
		<li>Inject 1-1.5 &#181;l DNA-solution containing 4 &#181;g DNA in the lateral ventricle (left or right)via the prepared borosilicate capillaries. Wait for 2-3 min, until the solution is diffused into the third ventricle. Alternatively directly inject into the third ventricle. 
		Note: This enhances specificity but might cause a decreased survival rate (high risk of damage).</li>
		<li>Apply voltage via the specialized forceps-type platinum electrodes (interval cycle length 50 msec, interval pause 950 msec).
		<ol>
			<li>Use a 0.5 or 3 mm electrode.</li>
			<li>Use a voltage ranging from 33-35 V.</li>
			<li>Place the center of the electrodes just posterior the ear primordia.</li>
			<li>To transfect the thalamus use angles from 25-40&#176;, to transfect the hypothalamus 90-180&#176; (Figure 2D).</li>
		</ol>
		</li>
		<li>Perform post electroporation/ post-operative care as described in section 1.4. Sacrifice the mice and analyze the embryos 6 days after electroporation (E18) as described in section 3.</li>
	</ol>
	</li>
</ol>
3. Perfusion Fixation
<ol>
	<li>Preparations
	<ol>
		<li>Warm 4% paraformaldehyde (PFA) to 37 &#176;C.</li>
		<li>Fill a 50 ml syringe with PBS (4 &#176;C) and a second syringe with warmed PFA. Avoid bubbles.</li>
		<li>Connect the syringes to a three-way stopcock. Connect the third end to a winged infusion set (butterfly).</li>
		<li>Flush the winged infusion set with PBS.</li>
	</ol>
	</li>
	<li>Perfusion
	<ol>
		<li>Deeply anesthetize the mice (pregnant or postnatal transfected) with subcutaneous application of ketamine hydrochloride (60 mg/ kg) and xylazine (7.5 mg/ kg).</li>
		<li>Confirm the surgical tolerance stage by unresponsiveness to toe-pinch.</li>
		<li>Open the abdominal cavity just beneath the rib cage.</li>
		<li>Carefully cut the diaphragm.</li>
		<li>Carefully open the rib cage via a cut on each site up to the collarbone.</li>
		<li>Insert the tip of the butterfly into the left heart chamber (starting from the apex of the heart).</li>
		<li>Cut the left atrial appendage.</li>
		<li>Slowly perfuse the mouse with PBS (approximately 10 ml).</li>
		<li>Switch the three-way stopcock.</li>
		<li>Perfuse the mouse with 5-10 ml 4% PFA.</li>
		<li>Decapitate the mouse using scissors and dissect the brain beginning at the foramen magnum. Perform the dissection as previously published 16.</li>
		<li>Postfixate the brain for 2 days in 4% PFA. Keep the brains at 4 &#176;C in the dark.</li>
	</ol>
	</li>
</ol>
4. Immunohistochemistry
<ol>
	<li>Generate 70 &#181;m sections (e.g., with a vibratome).</li>
	<li>Optional: Enhance fluorescence with antibody staining.
	<ol>
		<li>Block the sections for 1 hr at RT (0.1-0.2% Triton X-100, 5% normal goat serum)</li>
		<li>Incubate O/N with the appropriate antibody (anti-GFP, 1:1,000)</li>
		<li>Wash 3-5 times with 0.1 M phosphate buffer.</li>
		<li>Incubate 3-5 hr with the appropriate fluorescence-conjugated secondary antibody (Alexa Fluor 488 conjugated anti-rabbit-IgG, 1:1,000).</li>
		<li>Optional: Counterstain with 4-6-diaminodino-2-phenylindole (DAPI, 0.2 g/ ml in 0.1 M phosphate buffer) for 1 min.</li>
		<li>Analyze fluorescence with an appropriate fluorescence microscope.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This protocol describes in detail how to transfect 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 of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific r...

Since the first description in 2001 by three independent groups 1-3 in utero electroporation has become a widely used standard tool for analyzing gene expression in the rodent central nervous system. Compared to the generation of knockout mice, which is, despite continuously improving techniques, still time and money consuming, the in utero electroporation appeals due to its simplicity. So, in utero electroporation enables fast and efficient gain- and loss-of-function studies 4.
To transfect the cerebral regions, the solution containing the negatively charged plasmid is injected into a ventricle. During the electric pulse, the negatively charged DNA migrates towards the positive pole and therefore the transfected region can be selected simply by altering the position of the positive pole. It has frequently been shown that numerous regions of the central nervous system can be targeted 3,5-8. For instance, recent studies show specific transfections of the hippocampus, the piriform cortex or the striatum 9-11. However, the information about the appropriate positions are often only scarcely standardized and are not always easy to transfer to different mouse strains.
Transfection of certain embryonic stages is far from trivial. Many influencing factors must be taken into consideration when choosing the set-up for specific in utero electroporation. First, to optimally transfect the respective embryonic stages, knowledge about the appropriate voltages is needed. High voltages decrease the survival rate, whereas low voltages reduce the transfection efficiency 2,3,12. Also the size of the electrode paddle plays a crucial role, because the use of electrode paddles that are too large results in reduced specificity or can cause death due to affection of the heart rhythm 4,12,13. The applied voltage and the size and the position of the electrode paddle are the most important features to consider, but there are also further factors influencing the outcome of the electroporation, like the applied amount of DNA-solution.
We have developed a detailed protocol which enables fast and efficient transfection of various cerebral regions of the C57BL/6 mouse 12. In this protocol detailed information about the voltages to be used and the size of the electrode paddle for enhanced specificity is provided. Further, information about the ventricle to be filled along with recommendations for the amount of plasmid solution and the position of the electrode is supplied. The indication of the detailed position information in a map and the further visualization of these positions enables straightforward specific and efficient in utero electroporation of 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.

<table><tbody><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td></td></tr><tr><td>Fast Green</td><td>Roth</td><td>0301.1</td><td></td></tr><tr><td>pCAGGS</td><td>Addgene</td><td></td><td></td></tr><tr><td>borosilicate glass capillaries (0.8-0.9 mm diameter)</td><td>Wold Precision Instrument Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Isoflurane (Forene)</td><td>Abbott</td><td>PZN 4831850</td><td></td></tr><tr><td>Carprofen (Rimadyl)</td><td>Pfizer GmbH</td><td>approval number: 400684.00.00</td><td></td></tr><tr><td>eye ointment (Bepanthen Augen und Nasensalbe)</td><td>Bayer&amp;nbsp;</td><td>PZN 01578681</td><td></td></tr><tr><td>0.9% benzyl alcohol 0.9% saline solution</td><td>Pharmacy&lt;br/&gt; of the University Medical Center Mainz</td><td></td><td></td></tr><tr><td>gauze (ES-Kompressen)</td><td>Hartmann</td><td>407835</td><td></td></tr><tr><td>sterile 5-0 Perma-Hand Silk Suture</td><td>Ethicon Johnson &amp;amp; Johnson</td><td>K890H</td><td></td></tr><tr><td>ring forceps 1/ 1.5 mm</td><td>Fine Science Tools</td><td>11101-09</td><td></td></tr><tr><td>ring forceps 4.8/ 6 mm</td><td>Fine Science Tools</td><td>11106-09</td><td></td></tr><tr><td>ring forceps 2.2/ 3 mm</td><td>Fine Science Tools</td><td>11103-09</td><td></td></tr><tr><td>Adson Forceps-Serrated Straight 12 cm</td><td>Fine Science Tools</td><td>1106-12</td><td></td></tr><tr><td>IrisScissors-Delicate Straight-Sharp/Blunt 10 cm</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Mayo-Stille Scissors-Straight 15 cm</td><td>Fine Science Tools</td><td>14012-15</td><td></td></tr><tr><td>Dumont #5 Forceps-Inox</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm</td><td>Fine Science Tools</td><td>12565-14</td><td></td></tr><tr><td>Elektroporator CUY21 SC&amp;nbsp;</td><td>Nepa Gene Co.</td><td></td><td></td></tr><tr><td>FST 250 Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Microgrinder EG-44</td><td>Narishige</td><td></td><td></td></tr><tr><td>P-97 Micropette Puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Platinum electrodes 650P 0.5 mm</td><td>Nepagene</td><td>CUY650P0.5</td><td></td></tr><tr><td>Platinum electrodes 650P 3 mm</td><td>Nepagene</td><td>CUY650P3</td><td></td></tr><tr><td>Platinum electrodes 650P 5 mm</td><td>Nepagene</td><td>CUY650P5</td><td></td></tr><tr><td>Platinum electrodes 650P 10 mm</td><td>Nepagene</td><td>CUY650P10</td><td></td></tr><tr><td>Anesthesia system</td><td>Rothacher-Medical GmbH</td><td>CV-30511-3 Vapor 19.3</td><td></td></tr><tr><td>Heating plate</td><td>Rothacher-Medical GmbH</td><td>HP-1M</td><td></td></tr><tr><td>Temperature Controller 220V AC</td><td>Rothacher-Medical GmbH</td><td>TCAT-2LV</td><td></td></tr></tbody></table>

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.

Figure 2, shows examples for the specific in utero electroporation of the regions developing 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. The results of the transfections are shown next to the recommended angle (Figure 2). For better visualization of the angles in vivo the position of the electrode (0.5...

Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

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.

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 ...

cortex-hippocampus-thalamus-hypothalamus-lateral-septal-nucleus

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

27.9K Views.  University Medical Center, JGU Mainz. 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.

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

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

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

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 ...

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

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

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

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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 ...

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 ...

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

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

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

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

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

Summary

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Chapters in this video

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

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

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

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

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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