The authors would like to thank Jane Johnson for the Math1, Ngn1, and Ptf1a antibodies and Connie Cepko for the pCAG::GFP plasmid. This work was funded by NIH R15 1R15HD059922-01.

1. Preparations Prior to Electroporation
<ol>
 <li>Amplify the DNA for electroporation by a maxi prep (Prime-It or Qiagen). The concentration of the DNA should be a minimum of 1 mg/mL for efficient uptake.</li>
 <li>Remove 495 &mu;L of DNA and mix with 5 &mu;l of 0.01% Fast Green in 1 X PBS (phosphate buffered saline) in a microcentrifuge tube. </li>
</ol>
2. Embryonic Harvest
<ol>
 <li>Establish timed matings of CD-1 mice (Harlan). Check for the presence of vaginal plugs and regard the date a vaginal plug is observed as embryonic day (E) 0.5. Embryos will be harvested 11 days after visualizing plug (E11.5).</li>
 <li>Place sterilized tools in 70% ethanol. Treat a laminar flow hood with UV light for at least one hour prior to use. Spray hood, dissecting tray, and dissecting scope with 70% ethanol. Pre-heat 1 X PBS to 37 &deg;C.</li>
 <li>On E11.5 euthanize the female according to conditions approved by the Institutional Committee for the Care and Use of Animals (ICCUA). Place dissecting tray in the laminar flow hood. Spray down the abdomen of the female with 70% ethanol. </li>
 <li>Open the peritoneal cavity and pin the walls to the dissecting tray. Pull out the uterine horns so that it rests within the peritoneal cavity.</li>
 <li>Carefully cut open the wall of the uterine horn. Use a 20 mm spoon (Fine Scientific Tools) to gently remove the embryo in the yolk sac away from the placenta. Place embryos into a 100 mm tissue culture dish prefilled with 10 mL of sterile 1 X PBS that was preheated in 2.2.</li>
 <li>Repeat for all embryos present.</li>
</ol>
3. Electroporation of E11.5 Embryos (Figure 1)
<ol>
 <li>Using a 20 mm spoon, transfer the first embryo to a new 100 mm dish prefilled with 10 mL of sterile 1XPBS that was preheated to 37 &deg;C. </li>
 <li>Use 11 cm forceps with a 0.05 x 0.02 mm tip (Fine Scientific Tools) to carefully remove and discard the yolk sac.</li>
 <li>Position embryo so that its dorsal side is facing up so that it resembles the cartoon in Figure 1A. Use 7 mm electrode paddles (Harvard Apparatus) to gently hold the embryo. The electrodes should be positioned at either side of the neural tube at the level of the hindbrain fourth ventricle. The ventricle is visible to the naked eye but using a dissection microscope can facilitate accurate paddle placement. Positioning of the paddles is critical to determining the region that receives the electroporated DNA. If the lower rhombic lip (LRL) of the dorsal embryonic hindbrain is desired, paddles must be place such that they are directly flanking the widest part of the fourth ventricle opening.</li>
 <li>Use a 1 cc syringe to draw up the plasmid DNA mixed with 0.01% Fast Green. 500 &mu;L of the mixture should be sufficient for the electroporation of at least 8-10 embryos (see 3.5).</li>
 <li>Gently puncture the roofplate overlying the fourth ventricle with a 25G 5/8 tuberculin needle attached to the 1 cc syringe and inject the DNA-dye mixture into the ventricle. Successful injections are characterized by the DNA-dye mixture filling the entire ventricular system (Figure 1C). The amount of DNA-dye mixture typically injected is less than 50 &mu;L. Exact amounts are variable between embryos as a portion of the mixture tends to leak out of ventricle into the PBS surrounding the embryo. An alternate means of delivering the DNA-dye mixture would be through accessing the ventricular system by puncturing the velum overlying the midbrain. Again, successful injections are characterized by the DNA-dye mixture filling the entire ventricular system.</li>
 <li>Deliver five square pulses using an electric pulse generator (BTX) and 7 mm electrode paddles. Each pulse is 50 V lasting 5 ms per pulse with 500 ms between each pulse. The tissue closest to the positively charged electrode will then take up the plasmid.</li>
</ol>
4. Culture of Embryos
<ol>
 <li>In a laminar flow hood, fill the outer wells of a 12-well culture dish with 2 mL of DMEM/F12 media supplemented with 10% fetal bovine serum, 5% equine serum, 1% glutamine, 1% penicillin/streptomycin that was preheated to 37 &deg;C. The culture conditions were adapted from de Diego and colleagues.15</li>
 <li>In laminar flow hood pinch embryos at midsection (below the heart) with forceps and remove posterior portion of embryo. Place the anterior portion into one of the filled wells of the 12-well culture dish. </li>
 <li>Repeat for all embryos. Fill only the outer wells of the 12-well plate to avoid contamination of cultures.</li>
 <li>Culture embryos in a 37 &deg;C incubator with 5% CO2. Expression of the electroporated plasmid should be observable within 24 hr.</li>
 <li>Should longer culture times be desired, fill the out wells of a new 12 well plate with 2 mL of the media used in 4.1. With a sterilized spoon transfer the embryos to a well in the new plate. Place back into the 37 &deg;C incubator. Culture for up to 48 hr is possible.</li>
 <li>When the desired culture time is reached, fix embryos for analysis (see below). </li>
</ol>
5. Preparation of Embryos for Analysis
<ol>
 <li>Rinse embryos in 1 X PBS at 4 &deg;C for five min. Repeat.</li>
 <li>Fix embryos for analysis in 2% paraformaldehyde (PFA) in 1XPBS for 2 hours at 4 &deg;C.</li>
 <li>Rinse embryos in 1 X PBS at 4 &deg;C for five min. Repeat.</li>
 <li>Equilibrate embryos in 30% sucrose in 1 X PBS overnight at 4 &deg;C.</li>
 <li>Embed embryos in Optimal Cutting Temperature (O.C.T) compound using a dry ice/ethanol bath. Embryos can be stored at -20 &deg;C.</li>
 <li>Section embryos on a cryostat (Leica) into 30 &mu;m sections and mount on slides (VWR, Superfrost Plus). Store at -20 &deg;C.</li>
</ol>
6. Immunohistochemistry Analysis
<ol>
 <li>Immunohistochemistry was performed as described in 16. Primary antibody dilutions used for this study include rabbit &alpha;-GFP (Invitrogen) 1:2500; mouse &alpha;-Mash1 (BD Biosciences) 1:100; rabbit &alpha;-Ngn1 (Jane Johnson) 1:5000; rabbit &alpha;-Ptf1a (Jane Johnson) 1:2500; rabbit -Math1 (Jane Johnson) 1:100. Incubate slide flat, specimen side up on a staining tray at 4 &deg;C overnight.</li>
 <li>Slides were analyzed on a compound microscope (Olympus BX51).</li>
</ol>
7. Representative Results
A schematic in Figure 1A depicts the electroporation experiment. Figure 1B shows a sagittal view of an E11.5 embryo prior to manipulation. The same embryo following injection of the plasmid containing CAG::GFP in 0.01% Fast Green is shown in Figure 1C and a representative unfixed embryo exhibiting unilateral GFP expression in the dorsal hindbrain 24 hr following culture is shown in Figure 1D. The extent of the area of the LRL that is successfully electroporated is variable and appears to be highly dependent on positioning of the electrodes. In our studies it was found that 52 out of 65 (80%) of the electroporated embryos successfully expressed GFP. Tissue was deemed to be successfully electroporated if it was positive for GFP in localized regions over several sections following fixation and immunohistochemical analysis (see below). Embryos that failed to meet this criteria were scored as unsuccessful attempts at electroporation.
Further assessment of electroporation efficiency can be ascertained by performing immunohistochemistry against GFP on transverse sections of electroporated embryos at the level of the fourth ventricle. Figure 2 shows representative serial sections from an embryo displaying unilateral GFP expression. Figure 2A shows an idealized schematic that illustrates that the left side of the embryo was toward the positive electrode. Transverse sections (at levels represented by the middle cartoon of Figure 2A) reveal localized GFP expression exclusively on the left side of the hindbrain tissue (Figures 2C and 2D). The electroporated area of the LRL did not extend the entire anterior-posterior axis of the neural tube as examination of sections 300 &mu;m rostral or greater to that shown in Figure 2C does not express GFP (Figure 2B).
The utility of this assay for manipulation of gene expression is dependent upon the stability of the expression domains for the endogenous proteins. The LRL has been characterized as possessing unique progenitor domains characterized by the differential expression of proneural transcription factors (reviewed in 1). A subset of these factors (Mash1, Math1, Ngn1, and Ptf1a) were chosen for analysis due to their proposed and/or characterized roles in the specification of precerebellar neural subtypes in the LRL, a subject of future studies 16-18. All four proteins have highly characteristic expression domains in the caudal hindbrain at E11.5 16-18. We observed that embryos that were placed cultures failed to increase in size and also failed to initiate production of choroid plexus epithelium and invagination of the LRL and roofplate, morphological events that occur between E11.5 and E12.5 5. Based on these observations it was determined that normal development in these embryos was halted or grossly delayed and the comparable control for the cultured embryos should be uncultured embryos at E11.5.
To ensure that culture and electroporation do not disturb levels of endogenous proteins, we analyzed four different proteins by immunohistochemistry (IHC) in 34 different embryos that were electroporated and then cultured for at least 24 hr. Table I shows the number of embryos analyzed for each marker and the percentage of the embryos analyzed that retained normal protein levels. Figure 3 shows representative IHC data from two of the proteins analyzed, Mash1 (Figures 3A and 3B) and Math1 (Figures 3C and 3D). We observed that the majority of the embryos retained normal levels of expression following electroporation and culture (Figures 3B and 3D) as compared to the control embryos at E11.5 (Figures 3A and 3C). Importantly, the characteristic expression domains of these proteins were not perturbed.
<img src="/files/ftp_upload/3983/3983fig1.jpg" alt="Figure 1"/> 
Figure 1. Electroporation of Embryos at E11.5. (A) Schematic of the electroporation experiment. An E11.5 embryo is isolated and the expression plasmid in 0.01% Fast Green is injected into the fourth ventricle. The embryo is then flanked by electrode paddles and subjected to a 50 V pulse prior to being placed into culture (B) Sagittal view of an E11.5 embryo prior to injection. (C) The same E11.5 embryo following injection of plasmid in 0.01% Fast Green. (D) Unilateral hindbrain expression of GFP observed in E11.5 embryo following 24 hr of culture. mb- midbrain; 4v- fourth ventricle.
<img src="/files/ftp_upload/3983/3983fig2.jpg" alt="Figure 2"/> 
Figure 2. Expression of GFP in Electroporated Tissue. (A) The cartoon on left depicts the placement of electrodes around an E11.5 embryo. Middle cartoon depicts uptake and expression of plasmid encoding GFP on the left side of the embryo. Cartoon on right is schematic of an idealized transverse section taken through the embryo at levels denoted by black lines in middle cartoon. (B&#x2212;D) Immunohistochemistry for GFP on transverse sections through an electroporated E11.5 embryo after 24 hr of culture. Arrows indicate the roofplate (rp) which traps the secondary antibody. Images are taken at 10X magnification. The relative levels of the sections shown are depicted by the horizontal lines through the middle cartoon in (A). mb- midbrain;l 4v- fourth ventricle; rp- roofplate.
<img src="/files/ftp_upload/3983/3983fig3.jpg" alt="Figure 3"/> 
Figure 3. Expression of Endogenous Proteins in the Lower Rhombic Lip. Immunohistochemistry for Mash1 (A and B) or Math1 (C and D) comparing transverse sections of E11.5 embryos that were not cultured (A, C) with embryos that were electroporated with CAG::GFP and cultured for 24 hr (B and D). Images taken at 10X magnification.
<table border="1">
 <tbody>
 <tr>
 <td>Proneural Transcription Factor Analyzed</td>
 <td>Number of Embryos Analyzed</td>
 <td>Percentage Retaining Normal Expression Patterns</td>
 </tr>
 <tr>
 <td>Math1</td>
 <td>15</td>
 <td>86.7%</td>
 </tr>
 <tr>
 <td>Mash1</td>
 <td>12</td>
 <td>83.3%</td>
 </tr>
 <tr>
 <td>Ngn1</td>
 <td>7</td>
 <td>71.4%</td>
 </tr>
 <tr>
 <td>Ptf1a</td>
 <td>6</td>
 <td>100%</td>
 </tr>
 </tbody>
</table>
Table I. Percentage of Electroporated and Cultured Embryos Retaining Normal Proneural Transcription Factor Domains in the LRL.

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No conflicts of interest declared.

The in vitro electroporation technique presented in this study is a novel methodology that can be efficiently utilized to manipulate gene expression in embryos younger than 12 days of gestation. Placement of the embryos into culture permits expression of the introduced gene and circumvents the lethality observed when electroporated embryos are allowed to remain in vivo. This technique allows for the manipulation of gene expression in embryonic progenitors that were previously inaccessible for e...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Cryostat</td>
 <td>Leica</td>
 <td>CM-1850</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Biologie tip Dumoxel treated DUMONT forceps</td>
 <td>Fine Scientific Tools</td>
 <td>11252-30</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>20 mm MORIA perforated spoon</td>
 <td>Fine Scientific Tools</td>
 <td>10370-17</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ECM 830 Square Wave Electroporation Generator</td>
 <td>BTX (VWR)</td>
 <td>47745-928</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Harvard Apparatus 7 mm Tweezertrodes* Electrodes </td>
 <td>BTX (Fisher)</td>
 <td>BTX450165 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fisher Isotemp CO2 Incubator</td>
 <td>Fisher</td>
 <td>1325525 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NAPCO CO2 Gas Regulator</td>
 <td>Fisher</td>
 <td>15497020</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>12 Well Tissue Culture Plates</td>
 <td>BD Falcon (Fisher)</td>
 <td>877229</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HyClone Liquid Media DMEM/F-12 (1:1); With L-Glutamine and HEPES; 500mL </td>
 <td>Thermo Scientific (Fisher)</td>
 <td>SH3002301</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HyClone* Donor Equine Serum</td>
 <td>Thermo Scientific (Fisher)</td>
 <td>SH3007402 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum, Qualified, Heat Inactivated</td>
 <td>Invitrogen</td>
 <td>16140-063</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>cellgro* 10,000 IU Penicillin, 10,000&mu;g/mL Streptomycin </td>
 <td>Mediatech (Fisher)</td>
 <td>MT-30-002-CI </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HyClone* L-Glutamine L-Glutamine; 200mM in 0.85% NaCl </td>
 <td>Thermo Scientific (Fisher)</td>
 <td>SH3003401 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fast-Green</td>
 <td>Fisher</td>
 <td>AC41053-0250 </td>
 <td>0.01%</td>
 </tr>
 </tbody>
</table>

in vitro electroporation of the lower rhombic lip of midgestation mouse embryos

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle (reviewed in 1). The rhombic lip can be subdivided into the upper rhombic lip (URL) which encompasses rhombomere 1 (r1) and generates neurons of the cerebellum and the lower rhombic lip (LRL) which gives rise to diverse neuronal brainstem lineages 2-4. LRL derivatives include the auditory neurons of the cochlear nuclei and those of the precerebellar nuclei that are involved in regulating balance and motor control 5-8. Neurogenesis from the LRL occurs over a large temporal window that encompasses embryonic days (E) 9.5-16.55, 9. Different neuronal lineages emerge from the LRL as postmitotic cells (or are born) during distinct developmental days during this neurogenic window. 
Electroporation of gene expression constructs can be used to manipulate gene expression in LRL progenitors and can potentially change the fate of the neurons produced from this region 10-12. Altering gene expression of LRL progenitors in the mouse via in utero electroporation has been highly successful for manipulating lineages born on embryonic day E12.5 or later 10, 12-14. In utero electroporations prior to E12.5 have been unsuccessful primarily due to the lethality associated with puncturing the fourth ventricle roofplate, a necessary step in delivering exogenous DNA that is electroporated into the LRL. However, many LRL derived lineages arise from the LRL earlier than E12.5 9. These earlier born lineages include the neurons that comprise the lateral reticular, external cuneate, and inferior olivary nuclei of the precerebellar system which function to connect inputs from the spinal cord and cortex to the cerebellum 5. In order to manipulate expression in the LRL of embryos younger than E12.5, we developed an in vitro system in which embryos are placed into culture following electroporation.
This study presents an efficient and effective method for manipulating the gene expression of LRL progenitors at E11.5. Embryos electroporated with green fluorescent protein (GFP) driven from the broadly active CAG promoter reproducibly expressed GFP after 24 hours of culture. A critical aspect of this assay is that gene expression is only altered because of the expression of the exogenous gene and not because of secondary effects that result from the electroporation and culturing techniques. It was determined that the endogenous gene expression patterns remain undisturbed in electroporated and cultured embryos. This assay can be utilized to alter the fate of cells emerging from the LRL of embryos younger than E12.5 through the introduction of plasmids for overexpression or knock down (through RNAi) of different pro-neural transcription factors.

Watch this Scientific Journal Video about In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos at JoVE.com

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

This study describes the development of an in vitro electroporation technique that allows for the manipulation of gene expression in the lower rhombic lip of midgestation embryos.

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle ...

in-vitro-electroporation-lower-rhombic-lip-midgestation-mouse

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10.3K Views.  University of Illinois at Springfield. This study describes the development of an in vitro electroporation technique that allows for the manipulation of gene expression in the lower rhombic lip of midgestation embryos.

Video: In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

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 ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

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 ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.