This work was funded by NIH R01EY020560-01 and by a W.M. Keck Young Scholar in Medical Research Award. The authors would like to thank Joseph Bedont for his assistance during the imaging of retinal preparations and injections.

1. Plasmid preparation for electroporation
The DNA concentration required for electroporation is 5&mu;g/&mu;l. This typically requires the desired plasmids to be amplified using a Maxi-prep (Qiagen) or equivalent method followed by a purification and concentration of the DNA to the working amount. The following steps describe the preparation of DNA to the working amount.
<ol>
<li>Aliquot 100 &mu;g of DNA (from Maxi-prep or equivalent) and dilute the volume to 100 &mu;L for ease of manipulation.</li>
<li>Add phenol; the amount of phenol should be calculated to obtain an approximately 60% DNA/volume ratio (&mu;g DNA:total volume) i.e.: 100 &mu;g DNA (in 100 &mu;L) plus 67 &mu;L phenol (100 &mu;g:167 &mu;L). Mix thoroughly; do not pipette up and down as this may cause excessive shearing of the DNA.</li>
<li>Spin the DNA for 5 minutes at 14000 RPM at room temperature.</li>
<li>Collect supernatant and add chloroform using the same DNA:total volume ratio in step 1.2; mix as described in step 1.2 and spin at 14000 RPM for 5 minutes at room temperature.</li>
<li>Collect the supernatant, add 10 % supernatant volume of 3 M sodium acetate to the DNA solution, mix gently and add 2.5 X the supernatant volume of 100% ethanol. Mix by inversion. DNA should precipitate out of solution during the mixing.</li>
<li>Spin the DNA at 4&deg;C for 10 minutes at 14000 RPM.</li>
<li>Rinse with 350 &mu;l of 70 % ethanol, spin at 14000 RPM for 5 minutes at 4&deg;C.</li>
<li>Air dry the pellet then dissolve the DNA in 1 X PBS (Cell biology grade) to 5&mu;g/&mu;L. Do not overdry the sample as this will make it very difficult to dissolve into PBS.</li>
<li>Add Fast Green dye (10% stock) to the DNA solution, the final concentration of dye is 0.1%, to act as an injection tracer.</li>
</ol>
2. Subretinal injection of DNA
The following steps are performed with the assistance of a
stereomicroscope. Once practiced the process of eye opening, incision,
and injection takes less than 1.5 minutes which is not enough time for
a properly anesthetized pup to recover. Using a sharp 30-gauge needle,
carefully open the eye by cutting along the fused junctional
epithelium (Fig. 1C). Do not apply excessive force as this may result
in cutting of the underlying eye. Avoid cutting beyond the range of
the eyelid junction as this will result in bleeding which can obscure
the injection.
<ol>
<li>Anesthetize the newborn mice on ice for several minutes (Fig. 1A), do not bury pups in the ice as this may result in mortality. The
length of time on ice is variable from pup to pup, typically 5 minutes is sufficient but mice should be carefully monitored as individuals
may respond very quickly or may require longer exposure to ensure appropriate anesthesia. Conduct a paw pinch with hemostats to check
for withdrawal reflex.</li>
<li>In order to facilitate the injection of DNA to the subretinal space the eye must first be opened. Swab the eye to be injected with a 70% isopropyl alcohol prep (Tyco healthcare) and identify the fused junctional epithelium where the two eyelids come together (Fig. 1B).</li>
<li>Using a sharp 30-gauge needle, carefully open the eye by cutting along the fused junctional epithelium (Fig. 1C). Do not apply excessive force as this may result in cutting of the underlying eye. Avoid cutting beyond the range of the eyelid junction as this will result in bleeding which can obscure the injection.</li>
<li>To facilitate penetration of the eye by the blunt ended injection needle use the tip of a 30-guage needle make a small incision in the sclera near the junction with the cornea (Fig. 1E). Do not penetrate too deeply as this may result in a puncture of the lens.</li>
<li>Draw 0.3 &mu;l of DNA solution into a 33 gauge blunt ended injection needle using the syringe gradients to measure the volume individually for each injection (needle outer diameter 0.52 mm, inner diameter 0.13 mm; Exmire microsyringe; Ito corp). Insert the needle into the incision until the resistance of the opposing scleral wall is felt. Be careful to avoid penetrating the lens as the needle is passed through the vitreal chamber (Fig. 2B).</li> 
<li>Slowly inject 0.3 &mu;l of DNA solution into the subretinal space using the index finger or thumb to depress the syringe plunger. The experimenter must be careful to control the speed of plunger depression so that the rate at which the DNA solution is injected into the subretinal space does not outpace the rate at which the DNA solution spreads within subretinal space (Fig. 2C). The DNA solution is viscous so it is important that the needle is not pressed too tightly against the opposing scleral wall as this may result in the DNA not being injected or not spreading evenly. A successful injection will result in an even spread of DNA solution in a portion of the subretinal space. Regions of retina with and without underlying green tracer should be evident when rotating the injected animal.</li>
</ol>
3. Electroporation
<ol>
<li>Electroporation is performed using a 10 mm diameter tweezer electrode (Model #522; BTX Instruments). Soak the tweezer electrode in PBS in order to maximize electrical conductivity from the electrode to the animal and place the head of the injected pup between the electrodes with the injected eye adjacent to the positive pole electrode and the non-injected eye adjacent to the negative pole electrode (Fig. 3B).</li>
<li>Apply five square pulses using a pulse generator: each pulse is 80 volts and 50 milliseconds in duration with a 950 millisecond interval between pulses.</li>
<li>The electroporated pups must now be warmed until they recover from the ice anesthesia. This can be done by placing the pups under a warming lamp or on top of a slide warmer. If using a slide warmer ensure that an appropriate pad is placed between the metal surface and the recovering pup. Upon recovery return the electroporated pups to their mother.</li>
</ol>
4. Preparation of eyes for analysis
<ol>
<li>The time of harvesting and analysis of the electroporated retinas is performed subject to the objectives of the experiment. GFP expression can be grossly visualized 3 days after electroporation. For cryoembedding and sectioning proceed as follows.</li>
<li>Sacrifice electroporated animals at the desired timepoint. Disect out the electroporated eye and fix in 4% paraformaldehyde at 4&deg;C for 50 minutes.</li> 
<li>Carefully remove the retina from the eye by micro-dissecting away the sclera, cornea, lens, choroid and retinal pigmented epithelium. The dissected retinas may be analyzed under fluorescence to grossly determine the efficiency of electroporation. Transfer the retinas to 30% sucrose solution overnight at 4&deg;C.</li> 
<li>Freeze retinas into O.C.T. cryo-embedding media (Sakura Finetek USA) and store at -80&deg;C. Retinas may now be sectioned on a cryotome and captured onto glass slides.</li>
</ol>
5. Representative Results: 
Examples of P0 neonatal retinas electroporated with a pCAG-EGFP expression plasmid are presented at 3 days following electroporation (P3) and 14 days following electroporation (P14) in figure 4. The area of retinal tissue electroporated is variable from experiment to experiment depending on the extent of tissue damage incurred during the injection and evenness of the spread of the DNA solution into the subretinal space. Typically 90-100% of electroporated retinas will feature cells that have successfully incorporated and expressed the introduced plasmid. However, when factoring in tissue morphology and electroporation efficiency only 40-60 % of electroporated retinas will be suitable for comparative analysis. Rosette formation and retinal detachment at the site of needle penetration is nearly always observed and this region should not be used for analysis. Successful DNA spread during the microinjection results in efficiently electroporated tissue lateral to the site of needle penetration free from rosettes and detachment. Gliosis is also an important experimental concern and nearly always occurs at the site of needle penetration. However, as with rosette formation and retinal detachment, gliosis is typically not significant in laterally electroporated tissue. Immunohistochemical markers such as glial fibrillary acidic protein (GFAP) can be utilized to determine whether levels of reactive gliosis in analyzed regions fall within acceptable thresholds. Sectioned retinas demonstrating that the morphology of the electroporated retina remains intact and that EGFP expression labels individual electroporated cells are presented. Examples 3 days following a P0 electroporation (Fig. 4A-C) and 14 days following a P0 electroporation (Fig. 4D-F) are presented.
In P3 retinas the majority of electroporated cells are located in the neuroblastic layer (NBL) of the retina, and do not demonstrate distinct morphological characteristics of any of the differentiated neural or glial cells of the retina (Fig. 4B, C). By P14 electroporated cells can be found in the outer nuclear layer (ONL) and the inner nuclear layer (INL) of the retina. Furthermore the various labeled cells now display characteristic morphological hallmarks of differentiated neurons including interneurons of the INL, photoreceptors, and M&uuml;ller glia (Fig. 4E, F). 
<img src="/files/ftp_upload/2847/2847fig1.jpg" alt="Figure 1"/> 
Figure 1. Anesthesia of a newborn (P0) mouse pup and opening of eye for subretinal injection of DNA solution. A) Neonatal pup placed on a bed of crushed ice for anesthetization. Eye to be injected is placed down against the ice. B) The location of the fused junctional epithelium (B, arrow) of the eyelids to be cut for eye opening. C) Cutting of the eyelids along the fused junctional epithelium (C, arrow) to expose the eye. D) The opened eye following cutting at the fused junction. E) An incision is made into the eye beneath in the sclera beneath the cornea to facilitate easy insertion of the blunt ended micro-injection syringe. Scale bars, B, C, D, E: 4mm.
<img src="/files/ftp_upload/2847/2847fig2.jpg" alt="Figure 2"/> 
Figure 2. Insertion of the blunt ended micro-injection syringe and injection of DNA plasmid solution into the subretinal space. A) Cartoon schematic demonstrating the correct insertion of the micro injection syringe into the eye and settlement of the syringe tip into the subretinal space. Injection of the DNA solution into the vitreous chamber, passage of the syringe through the eye into the socket, or injection into the lens will result in failed experiments with few if any electroporated cells. B) Penetration of the syringe into the incision made in the scleral wall and settlement of the syringe tip into the subretinal space. C) Injection of the DNA solution into the subretinal space. Scale bars, B, C: 4mm. Abbreviations as follows: R-retina, L-lens, V-vitreous, SrS-subretinal space, BES-blunt ended syringe.
<img src="/files/ftp_upload/2847/2847fig3.jpg" alt="Figure 3"/> 
Figure 3. Orientation of the tweezer electrode onto the mouse for electroporation. A) Cartoon schematic demonstrating the orientation of the positive and negative paddles of the tweezer electrode relative to the electroporated eye. Green represents the location of injected DNA into the subretinal space. Dashed arrows represent the electrophoretic movement of negatively charged injected DNA towards the positive electrode. DNA electrophoresis occurs from the subretinal space adjacent to the negative electrode into the retina which is oriented towards the positive electrode. B) The positive paddle of the tweezer is placed adjacent to the DNA microinjected eye and the negative paddle is placed adjacent to the non-injected eye. C) High magnification image of tweezer electrode placement on the neonatal mouse. Dashed lines represent the direction of electrophoretic movement of DNA towards the positive electrode. Scale bar, C: 5mm.
<img src="/files/ftp_upload/2847/2847fig4.jpg" alt="Figure 4"/> 
Figure 4. Neonatal mouse retinas (P0) electroporated with pCAG-EGFP and analyzed at postnatal day 3 (P3) and postnatal day 14 (P14). A-C) Confocal images of a sectioned P3 retina electroporated with pCAG-EGFP at P0. The majority of electroporated cells sit in the retinal neuroblastic layer (NBL). (D-F) Confocal images of a sectioned P14 retina electroporated with pCAG-EGFP at P0. Electroporated cells can be identified in the outer nuclear layer (ONL) and the inner nuclear layer (INL) of the retina. Scale Bars, A-F: 50 &mu;m.

<ol>
	<li>Neumann, E., Rosenheck, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+changes+induced+by+electric+impulses+in+vesicular+membranes.">Permeability changes induced by electric impulses in vesicular membranes.</a> J. Membr. Biol. 10, 279-290 (1972).</li><li>Turnbull, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Letter:+Letter:+An+alternate+explanation+for+the+permeability+changes+induced+by+electrical+impulses+in+vesicular+membranes.">Letter: Letter: An alternate explanation for the permeability changes induced by electrical impulses in vesicular membranes.</a> J. Membr. Biol. 14, 193-196 (1973).</li><li>Zimmermann, U., Schulz, J., Pilwat, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcellular+ion+flow+in+Escherichia+coli+B+and+electrical+sizing+of+bacterias.">Transcellular ion flow in Escherichia coli B and electrical sizing of bacterias.</a> Biophys. J. 13, 1005-1013 (1973).</li><li>Kinosita, K., Tsong, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-induced+pore+formation+and+hemolysis+of+human+erythrocytes.">Voltage-induced pore formation and hemolysis of human erythrocytes.</a> Biochim. Biophys. Acta. 471, 227-242 (1977).</li><li>Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> EMBO J. 1, 841-845 (1982).</li><li>Swartz, M., Eberhart, J., Mastick, G. S., Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparking+new+frontiers:+using+in+vivo+electroporation+for+genetic+manipulations.">Sparking new frontiers: using in vivo electroporation for genetic manipulations.</a> Dev. Biol. 233, 13-21 (2001).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proc. Natl. Acad. Sci. U. S. A. 101, 16-22 (2004).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+expression+of+transgenes+introduced+by+in+vivo+electroporation.">Controlled expression of transgenes introduced by in vivo electroporation.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 1027-1032 (2007).</li><li>Onishi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pias3-dependent+SUMOylation+directs+rod+photoreceptor+development.">Pias3-dependent SUMOylation directs rod photoreceptor development.</a> Neuron. 61, 234-246 (2009).</li><li>Onishi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orphan+nuclear+hormone+receptor+ERRbeta+controls+rod+photoreceptor+survival.">The orphan nuclear hormone receptor ERRbeta controls rod photoreceptor survival.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 11579-11584 (2010).</li><li>Kim, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Core+Paired-Type+and+POU+Homeodomain-Containing+Transcription+Factor+Program+Drives+Retinal+Bipolar+Cell+Gene+Expression.">A Core Paired-Type and POU Homeodomain-Containing Transcription Factor Program Drives Retinal Bipolar Cell Gene Expression.</a> J. Neurosci. 28, 7748-7764 (2008).</li></ol>

No conflicts of interest declared.

In vivo electroporation represents a rapid and efficient method for
the transformation of retinal cells with DNA expression plasmids. This
method allows the experimenter to perform gain of function studies by
ectopically introducing a gene of interest under the control of a
ubiquitously expressed promoter or to perform loss of function studies
by using shRNA constructs targeting genes of interest. Furthermore,
multiple DNA plasmids can be electroporated simultaneously, allowing
the experimenter to analyze multip...

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of Reagent</th>
 <th>Company</th>
 <th>Catalogue Number</th>
 <th>Comments</th>
 </tr>
 <tr>
 	<td>Buffer Saturated Phenol</td>
 <td>Invitrogen</td>
 <td>15513-039</td>
 <td>N/A</td>
 </tr>
 	 <tr>
	 	<td>Chloroform</td>
 <td>J.T. Baker</td>
 <td>9180-03</td>
 <td>N/A</td>
 </tr>

 <tr>
 	<td>Sodium Acetate</td>
 <td>J.T. Baker</td>
 <td>3470-05</td>
 <td>3 M stock</td>
 </tr>

 <tr>
 	<td>Fast Green FCF</td>
 <td>Fisher Biotech</td>
 <td>BP123-10</td>
 <td>10 % stock </td>
 </tr>

 <tr>
 	<td>Isopropyl alcohol prep</td>
 <td>Tyco Healthcare</td>
 <td>6918</td>
 <td>N/A</td>
 </tr>
 <tr>
 	<td>30-guage needle</td>
 <td>Terumo Medical Corp.</td>
 <td>SG2-3013</td>
 <td>N/A</td>
 </tr>

 <tr>
 	<td>Exmire microsyringe</td>
 <td>Ito Corporation</td>
 <td>MS*E05</td>
 <td>N/A</td>
 </tr>

 <tr>
 	<td>Tweezertrode (tweezer electrode)</td>
 <td>BTX Instrument, Genetronics Inc.</td>
 <td>522</td>
 <td>N/A</td>
 </tr>
 <tr>
 	<td>Electro Square Porator (electroporator)</td>
 <td>BTX Instrument, Genetronics Inc.</td>
 <td>ECM 830</td>
 <td>N/A</td>
 </tr>
 <tr>
 	<td>O.C.T. Compound</td>
 <td>Sakura Finetek USA</td>
 <td>4583</td>
 <td>N/A</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about In vivo Electroporation of Developing Mouse Retina at JoVE.com

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.

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 ...

in-vivo-electroporation-of-developing-mouse-retina

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Neuroscience

20.7K Views.  Johns Hopkins School of Medicine. Метод включения плазмидной ДНК в мышиных клеток сетчатки для целей осуществления любой выигрыш или потеря функции исследования В естественных условиях Представлена. Этот метод капитализирует на кратковременное повышение проницаемости клеточных м?...

Video: In vivo Electroporation of Developing Mouse Retina

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 ...

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 ...

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 ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

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.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.