Name: gene delivery to postnatal rat brain by non-ventricular plasmid injection and electroporation
Uploaded: 2010-09-17T17:30:00
Description: Watch this Scientific Journal Video about Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation at JoVE.com

Wir danken Ekaterina Karelina für die Hilfe bei der Aufnahme Soundtrack für das Video, Ivan Molotkov für 3D-Animation und Dr. Peter Blaesse für CAG-EGFP Plasmid-Präparation. Die Arbeit wurde durch Zuschüsse aus dem Zentrum für Internationale Mobilität von Finnland, Finnish Cultural Foundation und der Academy of Finland unterstützt.

1. Einführung Erstellung von transgenen Tieren ist eine leistungsfähige Methode zur Untersuchung von Genfunktionen in lebenden Tieren 1 und zur Enträtselung Krankheitsmechanismus 2,3 sowie für die Manipulation von Eigenschaften der Zellen 4. Allerdings ist das Verfahren ziemlich aufwendig, extrem zeitaufwendig und teuer, so rechtfertigt die Verwendung von alternativen Gentransfer-Methoden wie virale Injektion 5, neonatalen ventrikulären Injektio...

<ol>
	<li>Gerlai, R., Clayton, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+hippocampal+function+in+transgenic+mice:+an+ethological+perspective.+Trends+Neurosci.">Analyzing hippocampal function in transgenic mice: an ethological perspective. Trends Neurosci.</a> 22, 47-51 (1999).</li><li>McGowan, E., Eriksen, J., Hutton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+modeling+Alzheimer's+disease+in+transgenic+mice.">A decade of modeling Alzheimer's disease in transgenic mice.</a> Trends Genet. 2, 281-289 (2006).</li><li>Cryan, J. F., Holmes, 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+ascent+of+mouse:+advances+in+modeling+human+depression+and+anxiety.">The ascent of mouse: advances in modeling human depression and anxiety.</a> Nat. Rev. Drug Discov. 4, 775-790 (2005).</li><li>Wells, T., Carter, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+neural+function+in+transgenic+rodents:+towards+a+comprehensive+strategy.">Genetic engineering of neural function in transgenic rodents: towards a comprehensive strategy.</a> J. Neurosci. Methods. 108, 111-130 (2001).</li><li>Pilpel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=reproducible+transduction+of+select+forebrain+regions+by+targeted+recombinant+virus+injection+into+the+neonatal+mouse+brain.">reproducible transduction of select forebrain regions by targeted recombinant virus injection into the neonatal mouse brain.</a> J. Neurosci. Methods. 182, 55-63 (2009).</li><li>Boutin, C. <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+vivo+electroporation+of+the+postnatal+rodent+forebrain.">Efficient in vivo electroporation of the postnatal rodent forebrain.</a> PLoS One. 3, e1883-e1883 (2008).</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, 237-246 (2001).</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. Protoc. 1, 1552-1558 (2006).</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>De Simoni, A., Yu, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+organotypic+hippocampal+slice+cultures:+interface+method.">Preparation of organotypic hippocampal slice cultures: interface method.</a> Nat. Protoc. 1, 1439-1445 (2006).</li><li>Walantus, W. <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+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> J. Vis. Exp. , (2007).</li><li>Walantus, W., Elias, L., Kriegstein, 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+intraventricular+injection+and+electroporation+of+E16+rat+embryos.">In utero intraventricular injection and electroporation of E16 rat embryos.</a> J. Vis. Exp. , (2007).</li><li>Umeshima, H., Hirano, T., Kengaku, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-based+nuclear+movement+occurs+independently+of+centrosome+positioning+in+migrating+neurons.">Microtubule-based nuclear movement occurs independently of centrosome positioning in migrating neurons.</a> Proc. Natl. Acad. Sci. U S A. 104, 16182-16187 (2007).</li><li>Ashwell, K., Paxinos, 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> Atlas of the Developing Rat Nervous System. , (2008).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (2007).</li></ol>

Methoden der Gentransfer in lebende Nagetier Gehirn sind gut für die in utero Elektroporation 7,8,11,12 gegründet und seit kurzem auch für die postnatale Elektroporation 6. Allerdings sind diese Methoden auf intraventrikuläre Injektion der Plasmid-DNA, die für mehrere Anwendungen kann die Begrenzung basiert. Zum Beispiel, haben diese Methoden nicht zulassen, dass Targeting-Zellen in bestimmten Bereichen des Gehirns wie Hippocampus, noch die Transfektion von solchen nicht-wandernde Zel...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2A-sa dumb Tweezers, 115mm</td>
<td>equipment</td>
<td>XYtronic</td>
<td>XY-2A-SA</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Biological Temperature Controller with stainless steel heating pad</td>
<td>equipment</td>
<td>Supertech</td>
<td>TMP-5b</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate tube with filament</td>
<td>material</td>
<td>Sutter Instruments</td>
<td>BF120-69-10</td>
<td>Glass needle</td>
</tr>
<tr>
<td>Disposable drills</td>
<td>material</td>
<td>Meisinger</td>
<td>HP 310 104 001 001 008</td>
<td>&nbsp;</td>
<tr>
<td>Dulbeco's PBS 10X</td>
<td>reagent</td>
<td>Sigma</td>
<td>D1408</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps, 110 mm</td>
<td>equipment</td>
<td>FST</td>
<td>91150-20</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Ealing microelectrode puller</td>
<td>equipment</td>
<td>Ealing</td>
<td>50-2013</td>
<td>Vertical electrode glass puller</td>
</tr>
<tr>
<td>Ethilon monofil polyamide 6-0 FS-3 16 mm 3/8c</td>
<td>material</td>
<td>Johnson &amp; Johnson Medical</td>
<td>EH7177H</td>
<td>Surgical threads</td>
</tr>
<tr>
<td>Exmire micro syringe 10.0 ml</td>
<td>equipment</td>
<td>Exmire</td>
<td>MS*GLLX00</td>
<td>Gas-tight syringe</td>
</tr>
<tr>
<td>Fast Green</td>
<td>reagent</td>
<td>Sigma</td>
<td>F7252</td>
<td>&nbsp;</td>
<tr>
<td>Forceps electrodes</td>
<td>equipment</td>
<td>BEX</td>
<td>LF650P3</td>
<td>Treat with 70% ethanol for disinfection prior to use</td>
</tr>
<tr>
<td>Foredom drill control</td>
<td>equipment</td>
<td>Foredom </td>
<td>FM3545</td>
<td>Surgical drill power supply and control. Currently available analogue is micromotor kit K.1070 (Foredom) </td>
</tr>
<tr>
<td>Foredom micro motor handpiece</td>
<td>equipment</td>
<td>Foredom</td>
<td>MH-145</td>
<td>Currently available analogue is micromotor kit K.1070 (Foredom)</td>
</tr>
<tr>
<td>Gas anesthesia platform for mice</td>
<td>equipment</td>
<td>Stoelting</td>
<td>50264</td>
<td>Assembled on stereotaxic instrument</td>
</tr>
<tr>
<td>Isoflurane</td>
<td>reagent</td>
<td>Baxter</td>
<td>FDG9623</td>
<td>&nbsp;</td>
<tr>
<td>Micro dressing forceps, 105 mm</td>
<td>equipment</td>
<td>Aesculap</td>
<td>BD302R</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Microfil</td>
<td>material</td>
<td>WPI</td>
<td>MF34G-5</td>
<td>Micro syringe filling capillaries</td>
</tr>
<tr>
<td>Mineral oil</td>
<td>reagent</td>
<td>Sigma</td>
<td>M8410</td>
<td>&nbsp;</td>
<tr>
<td>NanoFil Syringe 10 microliter</td>
<td>equipment</td>
<td>WPI</td>
<td>NANOFIL</td>
<td>Hamilton syringe</td>
</tr>
<tr>
<td>plasmid CAG-EGFP</td>
<td>reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Extracted and purified with EndoFree Plasmid Maxi Kit (Qiagen) and dissolved in nuclease free water to concentration 1.5 mg/ml</td>
</tr>
<tr>
<td>Pulse generator CUY21Vivo-SQ</td>
<td>equipment</td>
<td>BEX</td>
<td>CUY21Vivo-SQ</td>
<td>&nbsp;</td>
<tr>
<td>Schiller electrode gel</td>
<td>reagent</td>
<td>Schiller AG</td>
<td>2.158000</td>
<td>Conductive gel</td>
</tr>
<tr>
<td>Small animal stereotaxic instrument</td>
<td>equipment</td>
<td>David Kopf Instruments</td>
<td>900</td>
<td>&nbsp;</td>
<tr>
<td>Stoelting mouse and neonatal rat adaptor</td>
<td>equipment</td>
<td>Stoelting</td>
<td>51625</td>
<td>Assembled on stereotaxic instrument.
Treat earbars with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Student iris scissors, straight 11.5 cm</td>
<td>equipment</td>
<td>FST</td>
<td>91460-11</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Sugi absorbent swabs 17 x 8 mm</td>
<td>material</td>
<td>Kettenbach</td>
<td>31602</td>
<td>Surgical tampons</td>
</tr>
<tr>
<td>UMP3 microsyringe pump and Micro 4 microsyringe pump controller</td>
<td>equipment</td>
<td>WPI</td>
<td>UMP3-1</td>
<td>Microinjector and controller</td>
</tr>
<tr>
<td>Univentor 400 Anesthesia Unit</td>
<td>equipment</td>
<td>Univentor</td>
<td>8323001</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

gene delivery to postnatal rat brain by non-ventricular plasmid injection and electroporation

Erstellung von transgenen Tieren ist ein Standard-Ansatz bei der Untersuchung von Funktionen eines Gens von Interesse in vivo. Allerdings sind viele Knockout oder transgenen Tieren, die nicht in den Fällen, wo das modifizierte Gen exprimiert oder gelöscht wird in den gesamten Organismus lebensfähig. Darüber hinaus eine Vielzahl von Kompensationsmechanismen machen es oft schwierig, die Ergebnisse zu interpretieren. Die kompensatorische Effekte können entweder durch Timing der Genexpression oder die Begrenzung der Höhe von transfizierten Zellen vermindert werden. Die Methode der postnatalen nicht ventrikuläre Mikroinjektion und in vivo Elektroporation ermöglicht die gezielte Verabreichung von Genen, siRNA oder Farbstoffmoleküle direkt auf eine kleine Region des Interesses an der neugeborenen Nagern Gehirn. Im Gegensatz zu herkömmlichen ventrikuläre Injektionstechnik ermöglicht diese Methode die Transfektion von nicht-wandernde Zelltypen. Tiere mit Hilfe der hier beschriebenen Methode transfiziert kann, zum Beispiel für Zwei-Photonen werden in-vivo-Bildgebung oder in elektrophysiologischen Experimenten an akuten Hirnschnitten.

Watch this Scientific Journal Video about Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation at JoVE.com

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

Dieses Protokoll beschreibt eine nicht-virale Methode der Lieferung von Gen-Konstrukte zu einem bestimmten Bereich des Gehirns lebende Nagetier. Die Methode besteht aus Plasmid-Präparation, Mikropipette Fertigung, neugeborenen Ratten pup Chirurgie, Mikroinjektion des Konstrukts und<em&gt; In vivo</em&gt; Elektroporation.

Gene Lieferung in Postnatale Rattengehirn von Non-ventrikuläre Plasmid Injektion und Elektroporation

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

Research

JoVE Journal

Neuroscience

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 utero Elektroporation gefolgt von primären neuronalen Kultur für ein Studium Gene Function in Subset von kortikalen Neuronen

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

Forschung

Neurowissenschaften

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Mosaic Analyse der Genfunktion in Postnatale Mäusehirnen mithilfe von Virus-basierten Cre Rekombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Effiziente Gene Lieferung in mehrere ZNS Territories mit In Utero Elektroporation

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Maus in Utero Elektroporation: Kontrollierte Spatiotemporal Gene Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

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

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

Gentransfer an sich entwickelnden Maus Innenohr<em&gt; In Vivo</em&gt; Die Elektroporation

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 ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Erweiterung der embryonalen und adulten neuralen Stammzellen<em&gt; In Utero</em&gt; Elektroporation oder Viral stereotaktische Injektion

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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

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

In-vivo-Elektroporation und Postnatale Zeitraffer-Imaging Neuroblast von Migration in Maus Akute Hirnschnitten

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

Subtype-selective Electroporation of Cortical Interneurons

Subtyp-selektive Elektroporation kortikaler Inter

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

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

Die Analyse der Genexpression Veränderungen im Hippocampus Ratte nach Tiefenhirnstimulation des Anterior Thalamuskern

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

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...