This work was funded by RIKEN Brain Science Institute (BSI), Human Frontier Science Program (HFSP) (awarded to T.S.) and RIKEN Junior Research Associate Program (JRA, awarded to M.K.)RIKEN Brain Science Institute (BSI), Human Frontier Science Program (HFSP) (awarded to T.S.) and RIKEN Junior Research Associate Program (JRA, awarded to M.K.) funded this work.

1. Preparation of capillary and DNA solution for injection
<ol>
	<li>Purify plasmid DNA with a Maxi-prep or equivalent method, and make a final concentration of 2-3 &mu;g/&mu;l. Aliquot 100 &mu;g of DNA, add 10&mu;l of fast green dye (1% stock) and TE (10mM Tris Base, 1mM EDTA Solution, pH 8.0) and adjust a volume to 100 &mu;l to make the final DNA concentration for injection mix to 1&mu;g/&mu;l. The concentration of plasmid DNA can be changed depends on the size of plasmid, and also its transfection efficiency, which needs to be tested individually.</li>
	<li> Spin the DNA solution for 5 minutes at 14,000 RPM at room temperature and collect supernatant to remove any residue.</li>
	<li>Pull a glass capillary tubea glass capillary tubes using a micropipette puller. Pinch off the tip with forceps to make the tip has a 20&#x2013;30 &mu;m diameter. Measure 800 &mu;m-1 mm from the tip and check the external diameter is less than 60 &mu;m (Fig. 1A). </li> 
	<li>Connect the capillary to the micromanipulator, and fill with mineral oil. To fill the capillary with the DNA solution, submerge the tip into the solution and suck up the DNA solution. </li>
</ol>

2. Stick platinum electrodes 
Here we used stick platinum electrodes (Nepe gene, CUY611P2-1) to electroporate into superficial region of brain, such as the cortex (Fig. 1F). 

3. Preparation of micro-electrodes
Use micro-electrodes to electroporated into deep parts of the brain (i.e., thalamus and hypothalamus) (Fig. 2B-E). 

<ol>
	<li>One end of the tungsten (for negative electrodes) and platinum (for positive electrodes) wires is coiled on gold-plated pins and fixed with parafilm. Insert the wire to the stem of a cotton swab and wrap both ends with parafilm (Fig. 1B). </li>
	<li>Evenly sharpen the tip of the wire with sandpaper until it reaches 20&#x2013;30 &mu;m external diameter and 60 &mu;m from 800 &mu;m-1 mm of the tip (Fig. 1C and D).</li> 
	<li>Apply a thin coat of nail polish to the wire for insulation. After the nail polish has dried, use an acetone-soaked cotton swab to remove it from the tip (about 200 &mu;m from the tip). 
	Important note: In order to prevent damage to the mouse&#x2019;s uterus, the portion within 800 &mu;m-1 mm of the tip should be no more than 70 &mu;m in diameter (Fig. 1E). </li>
</ol>

4. Preparation of the surgery 
<ol>
	<li>Anesthetize a pregnant mouse (E9.5-E15.5). We demonstrate anesthetization with pharmaceutical grade sodium pentobarbital (Table II; 50 &mu;g per gram body weight, inject intraperitoneally and wait for 5 min). Alternatives to sodium pentobarbital as an anesthetic include Ketamine/Xylazine injection or gas anesthetics (gas is preferable for short duration procedures). The injection should be done with attention to avoid any organs, otherwise it will cause to damage the uterus or kill animal. When successful IP is done, the survival rate is more than 95%. Also the surgery can be performed in open-air environment. Before started the operation, test for a lack of response by the animal to pinching their toe to confirm anesthetic.</li>
	<li>Shave the hair from the abdomen using a razor blade and 50% EtOH. Prepare the skin with alternating scrubs of betadine and alcohol (70%).</li>
	<li>Make an incision at the abdominal midline with fine scissors and pull out all uterine horns carefully onto a 37 &deg;C pre-warmed phosphate-buffered saline (PBS)-moistened cotton gauze, which is placed around the wound. Keep the uterus moist with PBS all of the time.</li>
	<li>Hold a flexible fiber optic cable between the index and middle fingers and place it under the uterine horn. Clean cable with 70% EtOH prior to use. No microscope or magnifier is required for visualization. Position uterus between the optic fiber light optic cable and the thumb, and squeeze gently to push up the embryo closer to the uterine wall.</li>
</ol>

5. Injection of DNA and electroporation 
<ol>
	<li>When embryo is positioned, insert glass capillary carefully into target ventricle and inject approx 1 &mu;l of DNA solution (Fig. 2A).</li> 
	<li>For electroporation to superficial part of the brainIf using with stick platinum electrodes for electoporation, place the electrodes outside of the uterus and apply suggested square-wave current pulses according to manufacture&#x2019;s instruction (Table I). If usingFor electroporation to deeper part of the brain, nmicro electroporation, eedle type electrodes are used. Iinsert a fine tungsten negative electrode into DNA injected ventricle and a platinum electrode into the uterus and place target region between two electrodes, then apply suggested square-wave current pulses (Table I) (Fig. 2B).</li> 
</ol>

6. Post electroporation
<ol>
	<li>Place the uterine horn back in its original location with and add 500 &mu;L of 37 &deg;C pre-warmed PBS.</li>
	<li>Suture the inner layer with surgical suture and close outer layer with a 9 mm autoclipauto clip. </li>
	<li>Place animals on a heating pad for 2 hours to allow recovery from anesthesia. Analgesics such as carprofen or buprenorphine can be administered for post operative pain.</li>
</ol>
7. Representative results:
Some examples of cortical electroporation of pCAG-EYFP construct using stick platinum electrodes at different time points are shown in figure Figure 32. Brains are electroporated at E13.5, E14.5 and E15.5, and harvested at postnatal day (P) 6. AThe ntibody staining of RORc reveals position of layer 4 and position of EYFP transfected cells are revealed by GFP antibody staining, and both images are super imposed (Fig. 3A and B). Labeled cortical cells are layer of cortical cells electroporated is clearly different in each experiment (Fig. 32), which suggests time- dependent electroporation makes cortical layer-specific GOF/LOF possible. 
The viability of embryos and efficiency of cell transfection varies depending on the age of embryo and the location of electroporation. Samples are listed on Table 1, however, conditions should be optimized for each electroporation depending on the stage and part of the brain.
Electroporation into a deep tissue such as thalamus and hypothalamus with using stick platinum electrodes often give low efficiency (Table 1). This problem can be solved by using micro-electrodes whichelectrodes, which will reach to deep parts of the brain. Figure 3 4 shows example of pCAG-EYFP electroporation into the developing diencephalon. Plasmid DNA solution is injected into the 3rd ventricle at E11.5 and followed by micro electroporation (Fig. 3C4C). The electroporated area is restricted in the thalamus where most of the axons project to the cortex (Fig. 3B4B). Axon projection to layer 4 of the cortex can be also visualized by EYFP, which fillesfills up the entire neuron. (Fig. 3A4A).
<img src="/files/ftp_upload/3024/3024fig1.jpg" alt="Figure 1"/> Figure 1. Cartoon schematic demonstrating the correct shape of the capillary. (A) A glass capillary tube is pulled using a micropipette puller to make certain shape. A tip is pinched off by forceps to make it 20&#x2013;30 &mu;m. Measure 800 &mu;m-1mm from the tip (red bracket) and make sure that external diameter is less than 60 &mu;m (green bracket). (B) One end of the tungsten or platinum wires is coiled on gold-plated pins and fixed with parafilm. Then use sand paper to make their tip to become 20-30 &mu;m external diameters and 60 &mu;m from 1 mm of the tip. (C) Image of the tungsten wire before shaping. (D) Image of the tungsten wire after shaping. (E) Image of the tungsten wire after applying thin coat of nail polish. Scale bar in E is 10 &mu;m (smallest scale) for C-E. (F) Stick platinum electrodes (Nepa gene. CUY611P2-1). Scale bar in F is 2 mm.
<img src="/files/ftp_upload/3024/3024fig2.jpg" alt="Figure 2"/> Figure 2. Cartoon schema of surgical procedure. (A) Diagram of injection into lateral ventricle or large embryos (left side) and into 3rd ventricle or small embryos (right side). (B) Diagram of electrporation by platinum stick electrode (left side) and by needle type electrode (right side).
<img src="/files/ftp_upload/3024/3024fig3.jpg" alt="Figure 3"/> Figure 3. Developing mouse telencephalon was electroporated with pCAG-EYFP at E13.5 (A), E14.5 (B) and E15.5 (C) and harvested at P6. Brains were sectioned coronal and stained with GFP antibody or RORc antibody separately and images are merged. (A) Electroporation of pCAG-EYFP plasmid at E13.5 transfect many layer 4 neurons. which overlap with layer 4 markers RORc. (B) Electroporation of pCAG-EYFP plasmid at E14.5 transfect cells, which are more superficial than layer 4 neurons. (C) E15.5 electroporation label many superficial neurons such as layer 2/3.
Scale bar 200 &mu;m.
<img src="/files/ftp_upload/3024/3024fig4.jpg" alt="Figure 4"/> Figure 4. Transfection of EYFP into thalamo-cortical axons (TCA). (A) TCA terminus in cortical layer 4 is revealed by GFP antibody staining. RORc antibody staining is used to reveal a position of layer 4. Both images are taken from same section and merged. (B) Position of electroporation into the thalamus is also shown by GFP antibody staining and RORc staining, which expression is restricted in the thalamus 11. (C) Cartoon schematic for thalamic electroporation and TCA projection are shown. Scale bar 200 &mu;m.

<ol>
	<li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+Visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental Biology. 240, 237-246 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Kataoka, A., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf8+controls+regional+identity+in+the+developing+thalamus.">Fgf8 controls regional identity in the developing thalamus.</a> Development. 135, 2873-2881 (2008).</li><li>Imayoshi, I., Shimogori, T., Ohtsuka, T., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hes+genes+and+neurogenin+regulate+non-neural+versus+neural+fate+specification+in+the+dorsal+telencephalic+midline.">Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline.</a> Development. 135, 2531-2541 (2008).</li><li>Shimogori, T., Banuchi, V., Ng, H. Y., Strauss, J. B., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+signaling+centers+expressing+BMP,+WNT+and+FGF+proteins+interact+to+pattern+the+cerebral+cortex.">Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex.</a> Development. 131, 5639-5647 (2004).</li><li>Shimogori, T., Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+application+with+in+utero+electroporation+in+mouse+embryonic+brain.">Gene application with in utero electroporation in mouse embryonic brain.</a> Development Growth &#38; Differentiation. 50, 499-506 (2008).</li><li>Takahashi, Y., Watanabe, T., Nakagawa, S., Kawakami, K., Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+Embryology.">Avian Embryology.</a> Methods in Cell Biology. Vol 87, 271-271 (2008).</li><li>Feil, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-activated+site-specific+recombination+in+mice.">Ligand-activated site-specific recombination in mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 10887-10890 (1996).</li><li>Indra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally-controlled+site-specific+mutagenesis+in+the+basal+layer+of+the+epidermis:+comparison+of+the+recombinase+activity+of+the+tamoxifen-inducible+Cre-ERT+and+Cre-ERT2+recombinases.">Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases.</a> Nucleic Acids Research. 27, 4324-4327 (1999).</li><li>Nakagawa, Y., O'Leary, D. D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+patterned+expression+of+orphan+nuclear+receptor+genes+ROR+alpha+and+ROR+beta+in+developing+mouse+forebrain.">Dynamic patterned expression of orphan nuclear receptor genes ROR alpha and ROR beta in developing mouse forebrain.</a> Developmental Neuroscience. 25, 234-244 (2003).</li></ol>

No conflicts of interest declared.

In this protocol, we only described benefits of the technique of the pCAG-EYFP transfection into the restricted area of a small embryonic brain using different shaped electrodes. For comparison of transfection efficiency by different promoter is described previously, which shows cell type specificity 1. However, there are limitations for the technique, such as the transfection is only transient and varies depend on the plasmid, it does not have any cell specificity and it is hard to control numbers of transfected cells. ThereforeHowever, combining this protocol with other techniques will further provide possibilities for different manipulation methods. First, incorporating cell specific promoter into plasmid DNA will allow cell-type specific transfection, such as neurons, glial cells and astrocytes. Second, since the transfection is transient, it is not suitable for scientists who want to analyze the function of the gene in later life. Therefore, combination with a transposase to make plasmid DNA integrate into the genome will convert it into a stable transfection 8. Next, adoption of the tet-on or tet-off system will make it possible to manipulate the timing or cell type-specific gene expression in neural cells 8. Alternatively, one can combine this protocol with tamoxifen-inducible Cre-ER(T) recombinases 9 and reporter mice 10. 
This broad accessibility of tissues will drastically change experimental designs used in neuroscience.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>bare tungsten wire diameter 125 &mu;m</td>
			<td>A-M systems</td>
			<td>797600</td>
		</tr>
		<tr>
			<td>bare platinum wire diameter 125 &mu;m</td>
			<td>A-M systems</td>
			<td>767000</td>
		</tr>
		<tr>
			<td>Stick electrodes</td>
			<td>Nepa gene</td>
			<td>CUY610P4-1</td>
		</tr>
		<tr>
			<td>glass capillary tube</td>
			<td>Stoelting</td>
			<td>50611</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>KD Scientific</td>
			<td>KDS310</td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>ROBOZ</td>
			<td>RS-5865</td>
		</tr>
		<tr>
			<td>pulse generator</td>
			<td>A-M Systems</td>
			<td>Model 2100</td>
		</tr>
		<tr>
			<td>9 mm autoclip</td>
			<td>ROBOZ</td>
			<td>RS-9260</td>
		</tr>
		<tr>
			<td>gold-plated pins</td>
			<td>WPI	</td>
			<td>5482</td>
		</tr>
		<tr>
			<td>RORc antibody</td>
			<td>Perseus Proteomics</td>
			<td>H3925</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection at JoVE.com

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.

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 ...

mouse-utero-electroporation-controlled-spatiotemporal-gene

Research

JoVE Journal

Neuroscience

42.7K Views.  RIKEN Brain Science Institute. 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.

Video: Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

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 ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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 ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

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 ...

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

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

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

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

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

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

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

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

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

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

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