Name: induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models
Uploaded: 2015-01-12T12:30:00.000+00:00
Duration: 12 min 1 s
Description: 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

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><tbody><tr><td>ECM 830 Square Wave Electroporator</td><td>BTX Harvard Apparatus</td><td>45-0052</td><td>Electroporator only &amp;ndash; buy cables separately (45-0204)</td></tr><tr><td>1250FS Footswitch for ECM 830</td><td>BTX Harvard Apparatus</td><td>45-0211</td><td>Foot paddle is necessary for hands-free electroporation</td></tr><tr><td>Tweezertrodes</td><td>BTX Harvard Apparatus</td><td>45-0489 (5mm)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 45-0488 (7mm)</td><td>Platinum forcep style electrodes</td></tr><tr><td>Femtojet</td><td>Eppendorf</td><td>920010504</td><td>Microinjector</td></tr><tr><td>Griphead 0</td><td>Eppendorf</td><td>920007414</td><td>Holds the pulled needle</td></tr><tr><td>Universal Capillary Holder</td><td>Eppendorf</td><td>920007392</td><td>Connects griphead to tube</td></tr><tr><td>Injection Tube</td><td>Eppendorf</td><td>920007431</td><td>Connects capillary holder to Femtojet</td></tr><tr><td>Foot Control</td><td>Eppendorf</td><td>920005098</td><td>Foot paddle for hands-free injection</td></tr><tr><td>Microloader Tips</td><td>Eppendorf</td><td>930001007</td><td>Tips for loading the pulled capillary tubes</td></tr><tr><td>Plasmid Mega Kit</td><td>Qiagen</td><td>12181</td><td>5 QIAGEN-tip 2500, Reagents, Buffers</td></tr><tr><td>Fast Green FCF</td><td>Sigma-Aldrich</td><td>F7252-5G</td><td>Non-toxic dye</td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td>For trimming the needles</td></tr><tr><td>Bonn Strabismus Scissors</td><td>Fine Science Tools</td><td>14084-09</td><td></td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>11050-10</td><td>Straight</td></tr><tr><td>Colibri Retractors</td><td>Fine Science Tools</td><td>17000-03</td><td></td></tr><tr><td>Stapler</td><td>Fine Science Tools</td><td>12031-07</td><td>For 7mm clips</td></tr><tr><td>Castroviejo Needle Holder</td><td>Medical Tools</td><td>SNH-6737</td><td>Straight, with lock</td></tr><tr><td>Needle Puller</td><td>Narishige</td><td>PC-10</td><td>For pulling microcappillaries</td></tr><tr><td>Microcapillaries</td><td>Drummond</td><td>1-000-800</td><td>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 ...

induction-protein-deletion-through-utero-electroporation-to-define

Research

JoVE Journal

Developmental Biology

10.2K 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. 

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

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 ...

Neuroscience

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 ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

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

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

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

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

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

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

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, ...

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 ...

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

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

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

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

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

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

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

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