We are indebted to Pierre Mattar and Freda Miller for their assistance with the in utero electroporation and to Noel Ghanem and Bensun Fong for creating diagrams. We thank Linda Jui and Jason G. MacLaurin for excellent technical assistance. Equipment was supported by the Centre for Stroke Recovery.
This work was supported by a scholarship from HSFO and OGS to D.S.S. and a CIHR grant to R.S.S.

NOTE: All experiments were approved by the University of Ottawa&#8217;s Animal Care ethics committee, adhering to the Guidelines of the Canadian Council on Animal Care.
1. Pre-surgical Preparation and Materials
<ol>
	<li>Plasmid Preparation:
	<ol>
		<li>Prepare plasmids using the Qiagen MegaPrep according to the manufacturer&#8217;s protocol. Innoculate at least 400 ml of LB broth with transformed bacteria to ensure a high DNA yield and incubate overnight.</li>
		<li>Resuspend plasmid in 100-200 &#181;l TE to a final concentration of at least 5 &#181;g/&#181;l. Aliquot DNA and store at -20 &#176;C. If the concentration is too low, do not precipitate and resuspend as this can add impurities. Start over with a new preparation.</li>
		<li>Before surgery, dilute a single plasmid (Cre-GFP for example) to 2 &#181;g/&#181;l in sterile water with 1 &#181;l of 0.1% (w/v) Fast Green dye per 10 or 20 &#181;l of diluted plasmid solution. If GFP and the gene of interest are to be co-electroporated on separate plasmids, inject the GFP plasmid and the plasmid carrying the gene of interest at a ratio of 1:4 to maximize the likelihood that all cells marked with GFP are co-electroporated with both plasmids.</li>
	</ol>
	</li>
	<li>Pull glass microcapillary tubes (1 mm outer diameter - hereafter referred to as needles) on a standard needle puller, using a single step pull at 62 &#176;C.</li>
	<li>Autoclave a surgical pack containing the following: needle holders, wound retractor, forceps, scissors for surgery, scissors for needles, stapler, staples, gauze, wound covers, surgical drapes (Figure 1A, B; Materials Table).</li>
	<li>Collect other required tools: Electroporator, Femtojet Microinjecter, Tweezertrodes (5 or 7 mm), saline, suture, 5 ml syringe (Materials Table).</li>
</ol>
2. Prepare Mouse for Surgery
<ol>
	<li>For pain management, inject a timed-pregnant mouse with Buprenorphine (0.05 mg/kg body weight) one hour before surgery. Apply this protocol to mice timed anywhere from embryonic day 12.5 (E12.5) to E17.5.</li>
	<li>Use microloader pipette tips to back-load pulled needles with the plasmid solution. Do not fill all the way into the tip of needle yet as this will cause the needle to become clogged.</li>
	<li>Anaesthetize mouse using 2-5% isoflurane gas with 1 L/min O2. Once immobile, place in face mask and apply ointment to eyes to prevent drying. Inject with 1 ml saline (0.9% NaCl) subcutaneously in back. 
	NOTE: When working with isoflurane gas, use a gas scavenger to recapture excess gas and prevent it from escaping into the room.</li>
	<li>Shave abdomen. Wash abdomen with Chlorohexadine scrub, rinse with water, and apply a final prep solution of chlorohexadine and 70% ethanol. Cover mouse with sterile drape or gauze to cover fur but leave abdomen exposed (Figure 1C, D).</li>
</ol>
3. In utero Electroporation
<ol>
	<li>Make an incision in the skin over the abdomen between the lower teats. Pull the skin away from the underlying muscle layer by tearing the connective tissue, then cut through the peritoneum into the abdominal cavity along the linea alba. Insert the wound retractor to pull the edges of the wound apart.</li>
	<li>Remove the uterus from the abdominal cavity and lay it out as shown (Figure 1E).
	<ol>
		<li>If different pups are to be injected with different plasmids (such as GFP vs. GFP with the gene of interest), count the pups on each side and decide which ones and how many to inject with each plasmid. Replace one side of the uterus back into the abdomen to keep the embryos warm and lubricated. Immediately moisten the exposed uterus with saline (preferably warmed to body temperature).</li>
		<li>If injecting all pups with the same plasmid (such as when injecting Cre-GFP into transgenic embryos) only remove one horn of the uterus at a time.</li>
	</ol>
	</li>
	<li>Put a loaded needle in the grip head of the injection wand and carefully cut the tip with a pair of small surgical scissors or fine forceps. Cut the tip so the needle stays as long as possible while still allowing fluid to pass through (Figure 1Ji-iv).
	<ol>
		<li>Adjust length of injection (in seconds) and injection pressure on the microinjector so that a small yet easily visible droplet of 0.1-0.2 &#181;l appears on the tip of the needle when the foot paddle is pressed. Keep the volume of this droplet as consistent as possible between different needles. 
		NOTE: this step is crucial for embryo survival (see discussion).</li>
		<li>Use the following injection parameters for the Femtojet: injecting pressure (pi) = 250-300 hPa; injection time (ti) = 0.8-1.2 sec; compensation pressure = 10-50 hPa. If required, adjust these parameters to accommodate variation between needles.</li>
	</ol>
	</li>
	<li>Select an embryo and gently position it on its back, head tilted upward (Figure 1F). 
	NOTE: This may require turning or shifting the embryo; be careful not to apply too much pressure and avoid rupture of the amniotic sac while manipulating the embryo.</li>
	<li>Once the embryo is in place, locate the ventricle, which presents as a crescent shaped shadow parallel to the sagittal sinus. Touch the tip of the needle to the uterus above the ventricle at a slight angle and guide the needle through the uterine wall. The amount of pressure required depends on the sharpness of the needle. Push the needle through the cortex into the ventricle. 
	NOTE: Although there is rarely any additional detectable resistance, the embryo&#8217;s head is often pushed away slightly and then recoils as the needle passes through the cortex into the ventricle.</li>
	<li>Pump the foot paddle to inject the plasmid solution. Continue to pump until a green area is visible and conforms to the arc shape of the ventricle (Figure 1G). In young embryos (E12.5 to E14.5) the dye will often diffuse into the contralateral ventricle. 
	NOTE: The number of pumps depends on the diameter of the needle tip, the age of the embryo (and resulting size of the ventricle) and the desired injection volume. Keep volume injected as consistent as possible between embryos.</li>
	<li>Inject three or four embryos. Keep all exposed embryos moist with saline throughout procedure.</li>
	<li>Go back to the first embryos injected and place the paddles such that the positive electrode will draw the plasmid into the desired region of the brain (Figure 1H). Angle the negative electrode so that the shortest path between the electrodes passes directly through the structure targeted for electroporation (Figure 2).</li>
	<li>Once the paddles are in place, apply the shock. Depending on the age and size of the embryos, use 5 pulses of 35-50 V (see Table 1). Use the following electroporation parameters: pulse length of 5 msec with a pulse interval of 950 msec. Repeat with all injected embryos.</li>
	<li>Cut the tip of new pre-loaded needle as in step 3.3 as the last needle likely dried and clogged during step 3.9. Inject the next set of three or four embryos with either the same plasmid or a different plasmid and then apply the shock as in step 3.9. When all the pups in one side of the uterus are injected, remove the second side of the uterus from the female&#8217;s abdomen and then replace the completed side to keep it warm.</li>
	<li>Return both sides of the uterus to the female being careful to keep the surface moist with saline and not to apply too much pressure to prevent trauma to the amniotic sacs. Suture the muscle layer closed using a simple continuous stitch. Staple the skin closed. If staples are not available, suture the skin with a simple interrupted stitch. (Figure 1I)</li>
	<li>While suturing, progressively turn down the isoflurane to allow the pregnant female&#8217;s anesthesia to lighten. 
	NOTE: This will expedite arousal of the dam after the surgery is complete; keeping the female anesthetized for as short a time as possible will improve embryo survival. Total surgery time, starting from when the female is first anaesthetized, should be approximately 30 min.</li>
</ol>
4. Aftercare
<ol>
	<li>Place the sutured female in an incubator or on a heating pad to recover from anesthesia. Bed her on clean bedding and place a standard recovery gel in the cage maintain hydration.&#160;For pain management following surgery, give the female three doses of Buprenorphine (0.05 mg/kg body weight) at 8 hr&#160;intervals following surgery, then four additional doses at 12 hr&#160;intervals.&#160;</li>
</ol>
5. Harvesting Brains
NOTE: Brains can be harvested at any age up to adulthood. The following applies for collecting brains prior to birth. Note that once pups are born, the order within the uterus is lost, so either every pup in the litter must be injected with the same plasmid, or another method of differentiating between control and experimental animals must be devised.
<ol>
	<li>Prepare cold (4 &#176;C), filtered 4% paraformaldehyde (PFA) prior to euthanizing female.</li>
	<li>Euthanize the female using an intraperitoneal injection of a lethal dose of sodium pentobarbital (100 mg/kg body weight) and cut open abdomen. Pull out both sides of the uterus and lay out as when pups were initially injected.
	<ol>
		<li>In the case where all pups received the same plasmid, such as when injecting Cre into a transgenic litter, embryo order is not important, therefore cut the uterus out of the female and place in PBS.</li>
		<li>If pups were injected with different plasmids, be sure to locate each pup, noting any dead pups, and determine which ones were injected with control and experimental plasmids before removing the uterus from the female. Be careful to keep track of which pup belongs to which group after the uterus is in PBS.</li>
	</ol>
	</li>
	<li>Remove each pup individually, decapitate with sharp scissors or a razor blade and place the head in PBS. In the case of transgenic mice, collect tissue samples for genotyping and keep each head separate, such as in a 12-well plate.</li>
	<li>Under a dissecting scope, remove the brain from the skull, being careful not to nick the cortex or tear the midline. Using a fluorescent microscope, check to see if brains have a GFP+ patch, indicating that they were electroporated successfully.</li>
	<li>Fix brains by immersion in PFA at 4 &#176;C overnight. 
	NOTE: Check brains for expression of GFP prior to fixation since PFA often quenches the signal.</li>
	<li>Cryoprotect brains through immersion in 20% sucrose for at least three days prior to freezing. Use a sucrose gradient of 10%, 15% and 20% (24 hr in each solution) to prevent tissue damage due to osmotic pressure. Store frozen brains at -80 &#176;C and cut on a cryostat at 10-14 &#181;m thick sections.</li>
</ol>
6. Co-staining with anti-GFP
NOTE: If antigen retrieval is necessary in order to co-stain with anti-GFP, it is important to use a gentle antigen retrieval protocol that does not compromise the GFP signal. Some antigen retrieval protocols are too harsh to use in conjunction with GFP staining (even with an antibody against GFP). A basic citric acid pre-treatment prior to application of the primary antibody is successful for most epitopes 24.
<ol>
	<li>Make a 0.01 M solution of citric acid in ddH2O. pH to 6 using a calibrated pH meter.</li>
	<li>Heat citric acid in staining chamber in microwave or water bath until citric acids reaches 75-80 &#176;C. Put slides in staining chamber for 10-15 min. Maintain temperature as stable as possible.</li>
	<li>Wash slides in PBS 3x for 3 min at room temperature. Apply the primary antibody and proceed with immunostaining according to a standard protocol.</li>
	<li>Validate Cre mediated excision by co-labelling for GFP and the gene of interest to confirm knock down (Figure 3A).</li>
</ol>

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The authors have nothing to disclose

Two of the primary challenges in performing in utero electroporations are 1) preventing embryo lethality and 2) decreasing variability between electroporations. One of the most important factors in preventing lethality is needle quality, as blunt needles inflict more damage to the embryo. When cutting the needle, keep the tip as long as possible while still allowing a visible droplet of 0.1-0.2 &#181;l of fluid to appear following a pump of the foot paddle (Figure 1J). If the needle is too dull ...

Radial migration is a central process to early cortical development. It depends on a variety of cell autonomous factors, such as correct mitotic exit and neuronal differentiation, cell polarity, regulation of cytoskeletal dynamics and expression of transmembrane receptors, as well as non-cell autonomous factors, such as formation of the radial glial scaffold and secretion of migratory guidance molecules1-3. Since disruption of any of these mechanisms can impair neuronal migration in a transgenic mouse model4-8, determining the underlying cause of a defect in migration can be a complex and difficult process. In utero electroporation (IUE) can be used to complement and simplify interpretation of the phenotype of a genetic knock-out model and thereby elucidate important mechanisms required for radial migration7,9-11.
In utero electroporation (IUE) is the process by which a plasmid carrying both a gene of interest and a reporter is injected into the ventricle in the brain of a mouse or rat embryo and then drawn into the cells lining the ventricle through use of an electric current12-14. This allows the investigator to analyze the effect of up or down regulation of a gene of interest in development or function of electroporated neurons. Following IUE, brains can be processed for immunohistochemistry 7,9-11, electrophysiology 15 or cell culture 16,17. The major advantage of IUE is that is allows for highly specific manipulation of gene expression. Furthermore, IUE can be used to target a specific region of the developing brain through a directed current (Figure 2). IUE can also be used to target a specific cell type through injection of plasmids carrying genes under control of various promoters or plasmid activation systems (tetracycline induced gene expression is an example) 10,18-21.
IUE can be used in conjunction with the Cre-Lox mediated gene excision system 7-9,11,22. A plasmid containing a gene encoding the message for the Cre protein can be electroporated into an embryo homozygous for the floxed allele (in which the gene or specific DNA regions are flanked by two loxP sites for Cre mediated DNA recombination). Cre will then induce recombination of the gene of interest specifically in electroporated neural precursors, generating a knock-out of the floxed allele.The effect of protein knockdown on neural migration and development within individual neurons can then be studied. Electroporation of Cre induces recombination in only a small population of affected cells, leaving the supporting environment intact. In contrast, tissue specific expression of Cre under control of a cell type specific promoter, occurs throughout the entire tissue so that both migrating neuroblasts and the surrounding environment could be affected. Thus, juxtaposition of these two approaches can determine whether a given migration defect is due to cell autonomous or cell non-autonomous mechanisms. Defective migration in both experimental systems suggests that the observed phenotype results from a cell autonomous mechanism; normal migration following electroporation of Cre with defective migration in a tissue specific Cre model indicates that the gene of interest is acting through a non-cell autonomous mechanism.
IUE can also be used to perform rescue experiments by electroporating potential interacting genes into knock out animals 7,9,10. For example, an investigator could attempt to rescue a migration phenotype in a transgenic model by electroporating a downstream target and determining if the migration defect is corrected in the electroporated neurons. This has the additional benefit that a successful rescue indicates that normal migration can be restored by manipulating protein expression in a specific neuron, even though the surrounding environment is still deficient for the targeted gene. Again, this approach is more time and cost effective than crossing or generating transgenic lines, with the added benefit of determining whether the defective mechanism is cell autonomous.
IUE can be used to track migrating neurons through electroporation of a reporter plasmid into knock out embryos (Figure 4C). As only the neurons lining the ventricle at the time of surgery are electroporated 17, IUE can be used to follow the neurons born at a specific time with the advantage of visualization of their morphology in vivo.
Finally, it is possible to use IUE to target brain regions such as the medial or dorsal telencephalon, hippocampus or ganglionic eminence (Figure 2). This gives researchers the power to investigate gene function in a specific area independent of complications resulting from protein knockdown in neighboring structures.
Retinoblastoma protein (pRb), p107 and p130 comprise the pocket protein family and are well established regulators of cell cycle exit. However, there is increasing evidence that these proteins also regulate cell cycle independent aspects of neural development. As we have previously shown, pRb and p107 play a crucial role in both tangential 4,23 and radial migration 6. Here, we demonstrate the role of pocket protein family members pRb and p107 on neural migration 6 to exemplify the use of in utero electroporation of Cre in a transgenic model. In summary, IUE provides a powerful way of analyzing cell autonomous effects of gene deletion (or overexpression). When combined with tissue specific knock out models, IUE can provide additional information regarding the mechanisms controlling neural migration.

<table border="0" cellpadding="0" cellspacing="0" width="538">
	<tbody>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:233px;">Table 1: Equipment and Materials</td>
			<td style="width:64px;"></td>
			<td style="width:64px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:119px;">Product Name</td>
			<td style="width:115px;">Company</td>
			<td colspan="2" style="width:128px;">Catalog Number</td>
			<td style="width:177px;">Description</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:119px;">ECM 830 Square Wave Electroporator</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0052</td>
			<td style="width:177px;">Electroporator only &#8211; buy cables separately (45-0204)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">1250FS Footswitch for ECM 830</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0211</td>
			<td style="width:177px;">Foot paddle is necessary for hands-free electroporation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Tweezertrodes</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0489 (5mm)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 45-0488 (7mm)</td>
			<td style="width:177px;">Platinum forcep style electrodes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Femtojet</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920010504</td>
			<td style="width:177px;">Microinjector</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Griphead 0</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007414</td>
			<td style="width:177px;">Holds the pulled needle</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Universal Capillary Holder</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007392</td>
			<td style="width:177px;">Connects griphead to tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Injection Tube</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007431</td>
			<td style="width:177px;">Connects capillary holder to Femtojet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Foot Control</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920005098</td>
			<td style="width:177px;">Foot paddle for hands-free injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Microloader Tips</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">930001007</td>
			<td style="width:177px;">Tips for loading the pulled capillary tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Plasmid Mega Kit</td>
			<td style="width:115px;">Qiagen</td>
			<td colspan="2" style="width:128px;">12181</td>
			<td style="width:177px;">5 QIAGEN-tip 2500, Reagents, Buffers</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Fast Green FCF</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td colspan="2" style="width:128px;">F7252-5G</td>
			<td style="width:177px;">Non-toxic dye</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Extra Fine Bonn Scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">14084-08</td>
			<td style="width:177px;">For trimming the needles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Bonn Strabismus Scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">14084-09</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Graefe Forceps</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">11050-10</td>
			<td style="width:177px;">Straight</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Colibri Retractors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">17000-03</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Stapler</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">12031-07</td>
			<td style="width:177px;">For 7mm clips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Castroviejo Needle Holder</td>
			<td style="width:115px;">Medical Tools</td>
			<td colspan="2" style="width:128px;">SNH-6737</td>
			<td style="width:177px;">Straight, with lock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Needle Puller</td>
			<td style="width:115px;">Narishige</td>
			<td colspan="2" style="width:128px;">PC-10</td>
			<td style="width:177px;">For pulling microcappillaries</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Microcapillaries</td>
			<td style="width:115px;">Drummond</td>
			<td colspan="2" style="width:128px;">1-000-800</td>
			<td style="width:177px;">0.4 mm inner diameter glass capillary tubes</td>
		</tr>
	</tbody>
</table>

induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models

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

Different regions of the brain can be targeted for electroporation by varying the orientation of the paddle electrodes over the embryo&#8217;s head (Figure 2). For all parts of the forebrain, the plasmid is injected into the lateral ventricle (the hind brain can be targeted by injecting into the fourth ventricle). In general, place the electrodes so that the straight line between them passes through the region of interest, with DNA being drawn toward the positive electrode. To target the lateral cortex (...

Watch this Scientific Journal Video about Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models at JoVE.com

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

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

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

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

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Developmental Biology

10.1K Views.  University of Ottawa. Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment. 