Name: cortex-, hippocampus-, thalamus-, hypothalamus-, lateral septal nucleus- and striatum-specific in utero electroporation in the c57bl/6 mouse
Uploaded: 2016-01-18T14:00:00
Description: Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

Technical supported by Melanie Pfeifer and Nikolai Schmarowski (Institute for Microscopic Anatomy and Neurobiology, University Medical Center Mainz).

Ethics Statement: The handling of the mice and the experimental procedures were conducted in accordance with European, national and institutional guidelines for animal care.
1. In Utero Electroporation
Note: In utero electroporation was performed as previously published12,14. Therefore, the method is only described briefly in the following (Figure 1).
<ol>
	<li>Preparations
	<ol>
		<li>Prepare Fast Green colored endoto...

<ol>
	<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 (5544), 1071-1074 (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> Dev Biol. 240 (1), 237-246 (2001).</li><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 (4), 865-872 (2001).</li><li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex.">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cereb Cortex. 19 (July), i120-i125 (1991).</li><li>Nakahira, E., Yuasa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+generation,+migration,+and+differentiation+in+the+mouse+hippocampal+primoridium+as+revealed+by+enhanced+green+fluorescent+protein+gene+transfer+by+means+of+in+utero+electroporation.">Neuronal generation, migration, and differentiation in the mouse hippocampal primoridium as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation.</a> J Comp Neurol. 483 (3), 329-340 (2005).</li><li>Navarro-Quiroga, I., Chittajallu, R., Gallo, V., Haydar, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term,+selective+gene+expression+in+developing+and+adult+hippocampal+pyramidal+neurons+using+focal+in+utero+electroporation.">Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation.</a> J Neurosci. 27 (19), 5007-5011 (2007).</li><li>Borrell, V., Yoshimura, Y., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+delivery+to+telencephalic+inhibitory+neurons+by+directional+in+utero+electroporation.">Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation.</a> J Neurosci Methods. 143 (2), 151-158 (2005).</li><li>Kolk, S. M., de Mooij-Malsen, A. J., Martens, G. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+Molecular+Approach+of+in+utero+Electroporation+to+Functionally+Decipher+Endophenotypes+in+Neurodevelopmental+Disorders.">Spatiotemporal Molecular Approach of in utero Electroporation to Functionally Decipher Endophenotypes in Neurodevelopmental Disorders.</a> FNMOL. 4 (November), 37 (2011).</li><li>Tomita, K., Kubo, K. I., Ishii, K., 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=Disrupted-in-schizophrenia-1+(Disc1)+is+necessary+for+migration+of+the+pyramidal+neurons+during+mouse+hippocampal+development.">Disrupted-in-schizophrenia-1 (Disc1) is necessary for migration of the pyramidal neurons during mouse hippocampal development.</a> Hu Mol Gen. 20 (14), 2834-2845 (2011).</li><li>Niwa, M., Kamiya, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+DISC1+by+In+Utero+Gene+Transfer+Disturbs+Postnatal+Dopaminergic+Maturation+in+the+Frontal+Cortex+and+Leads+to+Adult+Behavioral+Deficits.">Knockdown of DISC1 by In Utero Gene Transfer Disturbs Postnatal Dopaminergic Maturation in the Frontal Cortex and Leads to Adult Behavioral Deficits.</a> Neuron. 65 (4), 480-489 (2010).</li><li>Nakahira, E., Kagawa, T., Shimizu, T., Goulding, M. D., Ikenaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+that+ventral+forebrain+cells+migrate+to+the+cortex+and+contribute+to+the+generation+of+cortical+myelinating+oligodendrocytes.">Direct evidence that ventral forebrain cells migrate to the cortex and contribute to the generation of cortical myelinating oligodendrocytes.</a> Dev Biol. 291 (1), 123-131 (2006).</li><li>Baumgart, J., Grebe, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C57BL/6-specific+conditions+for+efficient+in+utero+electroporation+of+the+central+nervous+system.">C57BL/6-specific conditions for efficient in utero electroporation of the central nervous system.</a> J Neurosci Methods. 240, 116-124 (2015).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat Protc. 1 (3), 1552-1558 (2006).</li><li>Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., 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=Mouse+in+utero+electroporation:+controlled+spatiotemporal+gene+transfection.">Mouse in utero electroporation: controlled spatiotemporal gene transfection.</a> JoVE. (54), (2011).</li><li>Guedel, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inhalation anesthesia: a fundamental guide. , (1937).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Animal+Perfusion+Fixation+for+Rodents.">Whole Animal Perfusion Fixation for Rodents.</a> JoVE. (65), e3564 (2012).</li><li>Mizutani, K., Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitors+resume+generating+neurons+after+temporary+inhibition+of+neurogenesis+by+Notch+activation+in+the+mammalian+cerebral+cortex.">Progenitors resume generating neurons after temporary inhibition of neurogenesis by Notch activation in the mammalian cerebral cortex.</a> Development. 132 (6), 1295-1304 (2005).</li><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=Labeling+embryonic+mouse+central+nervous+system+cells+by+in+utero+electroporation.">Labeling embryonic mouse central nervous system cells by in utero electroporation.</a> Dev, Growth &amp; Differ. 50 (6), 507-511 (2008).</li></ol>

This protocol describes in detail how to transfect 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 of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific r...

Since the first description in 2001 by three independent groups 1-3 in utero electroporation has become a widely used standard tool for analyzing gene expression in the rodent central nervous system. Compared to the generation of knockout mice, which is, despite continuously improving techniques, still time and money consuming, the in utero electroporation appeals due to its simplicity. So, in utero electroporation enables fast and efficient gain- and loss-of-function studies 4.
To transfect the cerebral regions, the solution containing the negatively charged plasmid is injected into a ventricle. During the electric pulse, the negatively charged DNA migrates towards the positive pole and therefore the transfected region can be selected simply by altering the position of the positive pole. It has frequently been shown that numerous regions of the central nervous system can be targeted 3,5-8. For instance, recent studies show specific transfections of the hippocampus, the piriform cortex or the striatum 9-11. However, the information about the appropriate positions are often only scarcely standardized and are not always easy to transfer to different mouse strains.
Transfection of certain embryonic stages is far from trivial. Many influencing factors must be taken into consideration when choosing the set-up for specific in utero electroporation. First, to optimally transfect the respective embryonic stages, knowledge about the appropriate voltages is needed. High voltages decrease the survival rate, whereas low voltages reduce the transfection efficiency 2,3,12. Also the size of the electrode paddle plays a crucial role, because the use of electrode paddles that are too large results in reduced specificity or can cause death due to affection of the heart rhythm 4,12,13. The applied voltage and the size and the position of the electrode paddle are the most important features to consider, but there are also further factors influencing the outcome of the electroporation, like the applied amount of DNA-solution.
We have developed a detailed protocol which enables fast and efficient transfection of various cerebral regions of the C57BL/6 mouse 12. In this protocol detailed information about the voltages to be used and the size of the electrode paddle for enhanced specificity is provided. Further, information about the ventricle to be filled along with recommendations for the amount of plasmid solution and the position of the electrode is supplied. The indication of the detailed position information in a map and the further visualization of these positions enables straightforward specific and efficient in utero electroporation of 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.

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:215px;">QIAGEN</td>
			<td style="width:188px;">12362</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green</td>
			<td>Roth</td>
			<td>0301.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCAGGS</td>
			<td>Addgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">borosilicate glass capillaries (0.8-0.9 mm diameter)</td>
			<td>Wold Precision Instrument Inc.</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane (Forene)</td>
			<td>Abbott</td>
			<td>PZN 4831850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carprofen (Rimadyl)</td>
			<td>Pfizer GmbH</td>
			<td>approval number: 400684.00.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eye ointment (Bepanthen Augen und Nasensalbe)</td>
			<td>Bayer&#160;</td>
			<td>PZN 01578681</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">0.9% benzyl alcohol 0.9% saline solution</td>
			<td style="width:215px;">Pharmacy 
			of the University Medical Center Mainz</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gauze (ES-Kompressen)</td>
			<td>Hartmann</td>
			<td>407835</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sterile 5-0 Perma-Hand Silk Suture</td>
			<td>Ethicon Johnson &#38; Johnson</td>
			<td>K890H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ring forceps 1/ 1.5 mm</td>
			<td>Fine Science Tools</td>
			<td>11101-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ring forceps 4.8/ 6 mm</td>
			<td>Fine Science Tools</td>
			<td>11106-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ring forceps 2.2/ 3 mm</td>
			<td>Fine Science Tools</td>
			<td>11103-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps-Serrated Straight 12 cm</td>
			<td>Fine Science Tools</td>
			<td>1106-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IrisScissors-Delicate Straight-Sharp/Blunt 10 cm</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mayo-Stille Scissors-Straight 15 cm</td>
			<td>Fine Science Tools</td>
			<td>14012-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Forceps-Inox</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elektroporator CUY21 SC&#160;</td>
			<td>Nepa Gene Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FST 250 Hot Bead Sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microgrinder EG-44</td>
			<td>Narishige</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P-97 Micropette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum electrodes 650P 0.5 mm</td>
			<td>Nepagene</td>
			<td>CUY650P0.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum electrodes 650P 3 mm</td>
			<td>Nepagene</td>
			<td>CUY650P3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum electrodes 650P 5 mm</td>
			<td>Nepagene</td>
			<td>CUY650P5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum electrodes 650P 10 mm</td>
			<td>Nepagene</td>
			<td>CUY650P10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthesia system</td>
			<td>Rothacher-Medical GmbH</td>
			<td>CV-30511-3 Vapor 19.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating plate</td>
			<td>Rothacher-Medical GmbH</td>
			<td>HP-1M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature Controller 220V AC</td>
			<td>Rothacher-Medical GmbH</td>
			<td>TCAT-2LV</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 2, shows examples for the specific in utero electroporation of the regions developing 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. The results of the transfections are shown next to the recommended angle (Figure 2). For better visualization of the angles in vivo the position of the electrode (0.5...

Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

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.

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 ...

Research

JoVE Journal

Neuroscience

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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 ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

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 ...

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

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 ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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 ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

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

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Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...