Name: efficient gene delivery into multiple cns territories using in utero electroporation
Uploaded: 2011-06-23T13:00:00
Description: Watch this Scientific Journal Video about Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation at JoVE.com

The authors would like to thank Eva Hadzimova, Pierre Mattar and Christopher Kovach for their initial work in establishing in utero electroporation technology in the CS lab. This work was funded by a Canadian Institute of Health Research (CIHR) grant (MOP 44094) and CIHR/Foundation Fighting Blindness (FFB) Emerging Team Grant (00933-000) to CS and an Alberta Children's Hospital Research Foundation Grant to DMK. RD was supported by a CIHR Canada Hope Scholarship, RC is supported by an FFB Studentship and LML was supported by a CIHR Training Grant in Genetics and Child Development.

1. Set-up
<ol>
	<li>Set-up surgical area as shown in Fig. 1A,A'. Key components of the set-up include an Eppendorf Femtojet microinjector, Narishige micromanipulator with needle holder, Leica steromicroscope, fiber optic lighting system, ECM 830 Square Wave Electroporation System with electrodes, heating pad and a vaporizer for isoflurane anesthetic.</li> 
	<li>Clean all tools with a Bransonic Ultrasonic Cleaner using Metriclean2 Low foaming solution for sonicating surgical tools. Sterilize tools by autoclaving. Steri...

<ol>
	<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, 237-246 (2001).</li><li>Takahashi, M., Sato, K., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+gene+expressions+by+electroporation+in+the+developing+brain+of+mammalian+embryos.">Manipulating gene expressions by electroporation in the developing brain of mammalian embryos.</a> Differentiation. 70, 155-162 (2002).</li><li>Langevin, L. 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Dev Dyn 236.</a> , 1273-1286 (2007).</li><li>Mattar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+helix-loop-helix+transcription+factors+cooperate+to+specify+a+cortical+projection+neuron+identity.">Basic helix-loop-helix transcription factors cooperate to specify a cortical projection neuron identity.</a> Mol Cell Biol. 28, 1456-1469 (2008).</li><li>Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-+and+loss-of-function+in+chick+embryos+by+electroporation.">Gain- and loss-of-function in chick embryos by electroporation.</a> Mech Dev. 121, 1137-1143 (2004).</li><li>Gaiano, N., Kohtz, J. D., Turnbull, D. H., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+rapid+gain-of-function+studies+in+the+mouse+embryonic+nervous+system.">A method for rapid gain-of-function studies in the mouse embryonic nervous system.</a> Nat Neurosci. 2, 812-819 (1999).</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>Petros, T. J., Rebsam, A., Mason, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+and+ex+vivo+electroporation+for+gene+expression+in+mouse+retinal+ganglion+cells.">In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells.</a> J Vis Exp. , (2009).</li><li>Garcia-Frigola, C., Carreres, M. I., Vegar, C., Herrera, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+into+mouse+retinal+ganglion+cells+by+in+utero+electroporation.">Gene delivery into mouse retinal ganglion cells by in utero electroporation.</a> BMC Dev Biol. 7, 103-103 (2007).</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>Vue, 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=Sonic+hedgehog+signaling+controls+thalamic+progenitor+identity+and+nuclei+specification+in+mice.">Sonic hedgehog signaling controls thalamic progenitor identity and nuclei specification in mice.</a> J Neurosci. 29, 4484-4497 (2009).</li><li>Tsuchiya, R., Takahashi, K., Liu, F. C., Takahashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+axonal+projections+from+mammillary+bodies+in+Pax6+mutant+mice:+possible+roles+of+Netrin-1+and+Slit+2+in+mammillary+projections.">Aberrant axonal projections from mammillary bodies in Pax6 mutant mice: possible roles of Netrin-1 and Slit 2 in mammillary projections.</a> J Neurosci Res. 87, 1620-1633 (2009).</li><li>Buffo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+pattern+of+the+transcription+factor+Olig2+in+response+to+brain+injuries:+implications+for+neuronal+repair.">Expression pattern of the transcription factor Olig2 in response to brain injuries: implications for neuronal repair.</a> Proc Natl Acad Sci U S A. 102, 18183-18188 (2005).</li></ol>

In utero electroporation can be used to analyze a wide variety of developmental processes. For example, transfection of reporter genes such as GFP, mCherry or alkaline phosphatase can be used to conduct lineage tracing and neuronal migration experiments. Alternatively, Cre recombinase can be transiently expressed to selectively eliminate a floxed allele in a spatially- and/or temporally-controlled manner. Furthermore, shRNA or dominant negative constructs can be electroporated to knockdown target gene function. ...

<table border="1">
	<tbody>
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 	<td>Ring forceps, 9mm</td>
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 	<td>Eye dressing Forcep</td>
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 	<td>Tissue forcep-Adson	</td>
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 	<td>Reflex Clip Applier</td>
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 	<td>Autoclip Remover</td>
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 	<td>Silk Black Braided Suture</td>
 <td>Ethicon Inc. </td>
 <td>K871</td>
 <td>Surgical Tools

</td>
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 	<td>Reflex Skin Closure Stainless Steel Wound Clips</td>
 <td>World Precision Instruments</td>
 <td>500346</td>
 <td>Surgical Tools</td>
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 <tr>
 	<td>ECM 830 Square Wave Electroporation System</td>
 <td>VWR-CanLab</td>
 <td>58018-004</td>
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 	<td>Tweezers w/Variable Gap 2 Round 5mm Platinum Plate Electrode</td>
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</td>
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 	<td>Tweezers w/Variable Gap 2 Round 7mm Platinum Plate Electrode

</td>
 <td>Protech International Inc.</td>
 <td>CUY650P7

</td>
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 	<td>Eppendorf Femtojet Microinjector</td>
 <td>VWR CanLab</td>
 <td>CA62111-488</td>
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 	<td>Foot Control for Eppendorf Femtojet Microinjector</td>
 <td>VWR CanLab</td>
 <td>CAACCESS (misc.)</td>
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 	<td>Bransonic Ultrasonic Cleaner 
Model 1510R-DTH</td>
 <td>VWR CanLab	</td>
 <td>CA33995-534
CPN-952-118

</td>
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 <tr>
 	<td>Sutter P97 Micropipet Puller </td>
 <td>Sutter Instrument,
 Carsen Group Inc.
</td>
 <td>P-97</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Micropipettes &#x2013; Borosilicate with filament 
O.D.: 1mm, I.D.: 0.78 mm, 10 cm length
</td>
 <td>Sutter Instrument</td>
 <td>BF100-78-10</td>
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 	<td>3-Axis Coarse Manipulator</td>
 <td>Carl Zeiss Canada Inc.</td>
 <td>M-152

</td>
 <td>Instruments</td>
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 <tr>
 	<td>Magnetic Holding Device for micromanipulator</td>
 <td>World Precision Instruments</td>
 <td>M1</td>
 <td>Instruments</td>
 </tr>
 <tr>
 	<td>Steel Base Plate for micromanipulator</td>
 <td>World Precision Instruments</td>
 <td>5052</td>
 <td>Instruments</td>
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 	<td>Micropipette Holder </td>
 <td>World Precision Instruments</td>
 <td>MPH3</td>
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 	<td>Micropipette Handle</td>
 <td>World Precision Instruments</td>
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</td>
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 	<td>Stereomicroscope</td>
 <td>Leica</td>
 <td>MZ6</td>
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 	<td>Vaporizer for isoflurane anesthetic

</td>
 <td>Porter Instruments Company

</td>
 <td>MODEL 100-F

</td>
 <td>Instruments</td>
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 	<td>Metriclean2 Low foaming solution for sonicating surgical tools</td>
 <td>Metrex Research Corporation</td>
 <td>10-8100</td>
 <td>Surgical Reagents</td>
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 	<td>Gentamicin 40mg/ml in 0.2 g methylene blue antibiotic spray after suturing</td>
 <td>Sigma Aldrich</td>
 <td>G1264</td>
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 	<td>Germex for sterilizing surgical tools</td>
 <td>V&eacute;toquinol</td>
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 <td>V&eacute;toquinol</td>
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 	<td>Sulpha &#x201C;25&#x201D; sulphamethazine oral antibiotic</td>
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 	<td>Lactated Ringer Solution</td>
 <td>Baxter Corporation</td>
 <td>DIN# 0061085</td>
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 <td>B-Braun Medical Inc.</td>
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 	<td>Inhalation Anesthetic &#x2013; Isoflurane USP</td>
 <td>Pharmaceutical
Partners of Canada Inc.</td>
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</td>
 <td>Surgical Reagents</td>
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 	<td>Fast Green FCF</td>
 <td>Sigma-Aldrich</td>
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 </tbody>
</table>

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.

Watch this Scientific Journal Video about Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation at JoVE.com

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.

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 ...

Research

JoVE Journal

Neuroscience

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

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

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 ...

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

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 ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

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

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 ...