Die Autoren danken die großzügige Unterstützung der James Madison University Department of Biology und James Madison Universität Licht Mikroskopie und Imaging Facility. Dr. Mark L. Gabriele für hilfreiche Ratschläge zur Vorbereitung junger postnatale Gewebe, und DRS. Justin W. Brown und Corey L. Cleland für großzügige Koordinierung der chirurgischen Materialien und Raum. Diese Forschung wurde teilweise durch ein Collaborative Research Grant durch 4-VA, eine partnerschaftliche Zusammenarbeit zur Förderung der Commonwealth von Virginia (etfolgreich) und durch eine Virginia Akademie der Wissenschaft kleine Projekt Research Grant (etfolgreich) finanziert. Unterstützung hat wurde großzügig zur Verfügung gestellt von einer Betty Jo liebevolle Butler 58 Stiftung für Undergraduate Research Stipendium (K.M.B), ein Sommer-Forschungsstipendium Farrell (auf K.M.B), ein James Madison Universität zweiten Jahrhundert Stipendium (K.M.B), ein James Madison University Centennial Stipendium (C.J.H.), ein James Madison Universität Lucy Robinson Suche 30 Memorial Stipendium (Z.L.H.) und ein James Madison University College von Wissenschaft und Mathematik Fakultät Hilfe gewähren (etfolgreich).

<ol>
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Die Autoren haben keine Interessenkonflikte offenlegen.

Hier stellen wir die Kombination von in Utero Elektroporation mit Cre-Rekombinase bei Floxed Mäusen, Mosaik Hirngewebe zu generieren. Ein Vorteil dieses Ansatzes ist, dass eine neue Mauslinie muss nicht jedes Mal generiert werden ein verschiedenen zellulärer Subtyp wird ins Visier genommen werden: in Utero Elektroporation kann verwendet werden, um exzitatorischen Neuronen, hemmende Neuronen oder Gliazellen je nach Ziel und Lage der Elektroporation15,<sup class="xref"...

Generierung von einem genetischen Mosaik ist ein klassischen experimentellen Paradigma zum Verständnis der Funktion eines Gens von Interesse. Um festzustellen, ob ein Gen für eine zelluläre Phänotyp notwendig ist, ist der einfachste Ansatz einen Verlust der Funktion des Gens in den Organismus (z.B. Ko) verursacht. Um festzustellen, ob ein Gen gezielt in einem bestimmten Zelltyp benötigt wird, ist jedoch nicht KO des Gens in den Organismus ein gültiger Ansatz. Stattdessen eine Methode ist erforderlich, dadurch wird den Verlust der Funktion eines Gens in einer bestimmten Zelle während von Wildtyp (d. h. genetisch unbeirrt) Gewebe umgeben ist – in anderen Worten, Mosaik Gewebe zu schaffen. Wenn die mutierte Zelle einen mutierten Phänotyp zeigt, aber umgebenden Wildtyp-Zellen nicht, der Genfunktionen Zelle-autonom. Analyse der Mosaik-Gewebe, in dem Wildtyp Gewebe, mutierte Zellen umgeben sind eignet sich für das Verständnis der Zelle-autonome Funktionen der Gene, vor allem im Gehirn, wo Neuronen und Glia eine große Verbundnetz des Gewebes bilden.
Verschiedene Formen von Mosaik Hirngewebe lieferten leistungsstarke Modelle um Zelle-autonome Funktionen der Gene zu untersuchen. Studien konzentrierten sich auf neuronale Transplantation1weiblichen X-chromosomal Mosaikbildung2,3,4, und endogenen somatische Mosaikbildung5,6 haben ihre Schlußfolgerungen basierend auf Mosaik Hirngewebe. Bedingte Löschung eines Gens durch die Cre Lox Rekombinationssystem ist eine Methode, die die große Verfügbarkeit der transgene Mauslinien nutzt. Bei dieser Methode werden zwei LoxP-Standorten eingeführt, auf beiden Seiten einer erforderlichen Sequenz eines Gens (z. B. ein Exon), flankiert von LoxP-Websites, die beide in die gleiche Richtung ("Floxed") stehen lassen. Cre-Rekombinase beschneidet die Reihenfolge zwischen den LoxP-Websites-7. CRE-vermittelten Rekombination kann durch Kreuzung Floxed Mäuse mit einer anderen Mauslinie mit dem Ausdruck Cre-Rekombinase zusammen mit einem fluoreszierenden Marker in einer Teilmenge von Zellen ("Cre-Reporter-Linie") erreicht werden. Dies wurde in einer Vielzahl von Möglichkeiten, um die Funktionen eines Gens in Teilmengen von Zellen, wie z. B. exzitatorischen Neuronen oder Astrozyten8aufzudecken nachgewiesen. CRE-Reporter Linien CreERT2 Cre-vermittelten Rekombination sein Medikament-induzierbaren (Single Neuron Etikettierung mit induzierbaren Cre-vermittelten Ko oder glatt) ermöglichen ausdrücken können9. In eine andere Strategie als Mosaik-Analyse mit doppelter Marker (MADM)10,11ermöglicht Cre-vermittelten interchromosomal Rekombination eine homozygote Mutante neben heterozygot Gewebe geschaffen werden. In diesen Ansätzen muss eine neue Linie von Mäusen produziert werden jedes Mal für jeden Kandidaten-gen oder zellulären Subtyp, der überprüft wird. Alternativ kann Cre-Rekombinase postnatal durch Iontophorese12 oder durch virale Vektoren (z.B. Adeno-assoziierten Viren13 oder Lentiviren14 mit zellulären Subtyp-spezifische eingeführt werden Promotoren). Diese Strategie schafft starke und postnatale beschriften. Zu dünn und pränatal zerebrale kortikale Neuronen entwickeln, ist eine ideale Strategie in Utero Elektroporation Cre-Rekombinase mit einem fluoreszierenden Marker.
Neben der Kombination von Cre Lox Rekombination durch in Utero Elektroporation produzieren Mosaik Gewebe in Vivo, mehrere Anpassungen stellen wir Verfahren aus anderen veröffentlichten Protokolle15,16, 17,18,19,20,21. Wir geben Informationen zum Erfolg in der Zucht timed-trächtige Weibchen zu verbessern. Auch beschreiben wir unsere zwei Strategien einzuführen, spärlich und hellen Kennzeichnung von Neuronen im kortikalen Gewebe: eine Strategie ist, die Ebenen von einem einzigen Konstrukt Codierung für Cre-Rekombinase und einem fluoreszierenden Marker22titrieren. Eine weitere Strategie ist das "Supernova"-System, speziell mit diesen Parametern im Geist23,24verwenden. Darüber hinaus bieten wir Verbesserungen auf die Herstellung von konsistenten Mikroinjektion Pipetten und Vereinfachungen, die in Utero Elektroporation Chirurgie. Zu guter Letzt erläutern wir wichtige Schritte in einem vereinfachten histologischen Präparat, das die Analyse von dendritischen Dornen und dendritischen und axonalen Dorne, ohne weitere Färbung oder Immunohistochemistry ermöglicht.

<table><tbody><tr><td>C57BL/6J mice</td><td>The Jackson Laboratory</td><td>#000664</td><td>See &quot;1. Mouse set-up&quot; (step 1.1, &quot;wildtype mice&quot;)</td></tr><tr><td>GFP.Cre empty vector</td><td>AddGene</td><td>#20781</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;single DNA construct that codes for Cre recombinase as well as a fluorescent marker&quot;). GFP.Cre empty vector was a gift from Tyler Jacks.</td></tr><tr><td>pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (Supernova)</td><td>AddGene</td><td>#69138</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; system) and http://snsupport.webcrow.jp/. pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (Supernova) was a gift from Takuji Iwasato.</td></tr><tr><td>pK031.TRE-Cre (Supernova)</td><td>AddGene</td><td>#69136</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; system) and http://snsupport.webcrow.jp/. pK031.TRE-Cre (Supernova) was a gift from Takuji Iwasato.</td></tr><tr><td>pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova)</td><td>AddGene</td><td>#85006</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; system) and http://snsupport.webcrow.jp/. pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova) was a gift from Takuji Iwasato.</td></tr><tr><td>EndoFree Plasmid Maxi Kit (10)</td><td>Qiagen</td><td>#12362</td><td>See &quot;2. DNA set-up&quot; (step 2.3 &quot;endotoxin-free plasmid purification kit&quot;)</td></tr><tr><td>Trypan Blue powder, BioReagent grade</td><td>Sigma</td><td>T6146-5G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;trypan blue&quot;)</td></tr><tr><td>Sodium Chloride, ACS, 2.5 kg</td><td>VWR</td><td>BDH9286-2.5KG</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;NaCl&quot;)</td></tr><tr><td>Potassium Chloride, ACS, 500 g</td><td>VWR</td><td>#97061-566</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;KCl&quot;)</td></tr><tr><td>Sodium phosphate dibasic, ReagentPlus, 100 g</td><td>Sigma-Aldrich</td><td>S0876-100G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&quot;)</td></tr><tr><td>Potassium phosphate monobasic, ReagentPlus, 100 g</td><td>Sigma-Aldrich</td><td>P5379-100G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&quot;)</td></tr><tr><td>Hydrochloric acid, ACS reagent, 500 mL</td><td>Fisher Scientific</td><td>A144-500</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;HCl&quot;)</td></tr><tr><td>P-97 Micropipette Puller</td><td>Sutter Instrument</td><td>P-97</td><td>See &quot;3. Pipette set-up&quot; (step 3.1 &quot;glass capillary puller&quot;)</td></tr><tr><td>3.0 mm wide trough filament</td><td>Sutter Instrument</td><td>FT330B</td><td>See &quot;3. Pipette set-up&quot; (step 3.1 &quot;glass capillary puller&quot;)</td></tr><tr><td>Thin Wall Glass Capillaries, 4&quot;, 1 / 0.75 OD/ID</td><td>World Precision Instruments</td><td>TW100-4</td><td>See &quot;3. Pipette set-up&quot; (step 3.1.1 &quot;glass capillary&quot;)</td></tr><tr><td>Single Ply Soft-Tech Wipes, 4.5&quot;</td><td>Phenix</td><td>LW-8148</td><td>See &quot;3. Pipette set-up&quot; (step 3.2.1 &quot;single-ply task wipe&quot;); other single-ply wipes (e.g. Kimwipes) can be used.</td></tr><tr><td>Graefe Forceps, 7 cm, Straight, 0.7 mm 1x2 Teeth</td><td>World Precision Instruments</td><td>#14140</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Graefe forceps&quot;)</td></tr><tr><td>Iris Scissors, 11.5 cm, Straight, 12-pack</td><td>World Precision Instruments</td><td>#503708-12</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;iris scissors&quot;)</td></tr><tr><td>Hartman Mosquito Forceps, 9 cm, Straight, 12-pack</td><td>World Precision Instruments</td><td>#503728-12</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Hartman mosquito forceps&quot;)</td></tr><tr><td>General Purpose Non-Woven Sponges, 2&quot; x 2&quot;, 4-ply</td><td>Medrepexpress</td><td>#2204-c</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;non-woven gauze sponges&quot;)</td></tr><tr><td>Ring Tipped Forceps, 10 cm, Straight, 2.2mm ID</td><td>World Precision Instruments</td><td>#503203</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;ring-tipped forceps&quot;)</td></tr><tr><td>Pyrex petri dishes complete, O.D. &amp;times; H 100 mm &amp;times; 20 mm</td><td>Sigma-Aldrich</td><td>CLS3160102-12EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Petri dishes&quot;)</td></tr><tr><td>Flat Type Instrument Tray, Stainless Steel, 13-5/8&quot; x 9-3/4&quot; x 5/8&quot;</td><td>Amazon</td><td>B007SHGAHA</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;stainless steel tray&quot;)</td></tr><tr><td>Platinum Tweezertrode, 5 mm</td><td>BTX</td><td>#45-0489</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 and 4.16 &quot;tweezer-type electrodes&quot;)</td></tr><tr><td>ECM 830 Foot Pedal</td><td>BTX</td><td>#45-0211</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 and 4.17 &quot;foot pedal&quot;)</td></tr><tr><td>ECM 830 Generator</td><td>BTX</td><td>#45-0052</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 &quot;generator&quot;)</td></tr><tr><td>Single Animal Isoflurane Anesthesia System with Small Induction Box</td><td>Harvard Apparatus</td><td>#72-6468</td><td>See &quot;4. In utero electroporation&quot; (step 4.4 and 4.6 &quot;nose cone&quot;, step 4.4 &quot;induction chamber&quot;)</td></tr><tr><td>Ophthalmic ointment</td><td>Hanna Pharmaceutical Supply Co</td><td>#0536108691</td><td>See &quot;4. In utero electroporation&quot; (step 4.7 &quot;veterinary ophthalmic ointment&quot;)</td></tr><tr><td>Space Gel (AIMS)</td><td>VWR</td><td>#95059-640</td><td>See &quot;4. In utero electroporation&quot; (step 4.8 &quot;sealed pouch filled with supersaturated salt solution&quot;)</td></tr><tr><td>Hair Remover Gel Cream, Sensitive Formula</td><td>Veet</td><td>#062200809951</td><td>See &quot;4. In utero electroporation&quot; (step 4.9 &quot;depilatory cream&quot;)</td></tr><tr><td>10ul Low Retention Tip Starter (960 tips/pk)</td><td>Phenix Research Products</td><td>TSP-10LKIT</td><td>See &quot;4. In utero electroporation&quot; (step 4.12 &quot;sterile 10 &amp;micro;L micropipette tip&quot;)</td></tr><tr><td>Aspirator tube assemblies for calibrated microcapillary pipettes</td><td>Sigma-Aldrich</td><td>A5177</td><td>See &quot;4. In utero electroporation&quot; (step 4.15 &quot;aspirator tube assembly&quot;)</td></tr><tr><td>Braided Absorbable Suture, 4-0, Needle NFS-2(FS-2), 27&quot;</td><td>Medrepexpress</td><td>MV-J397</td><td>See &quot;4. In utero electroporation&quot; (step 4.19 &quot;absorbable sutures&quot;)</td></tr><tr><td>&amp;ldquo;LiquiVet Rapid&amp;rdquo; Tissue Adhesive</td><td>Medrepexpress</td><td>VG3</td><td>See &quot;4. In utero electroporation&quot; (step 4.20 &quot;tissue adhesive&quot;)</td></tr><tr><td>Hypodermic syringes, polypropylene, Luer lock tip, capacity 1.0 mL</td><td>Sigma-Aldrich</td><td>Z551546-100EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.21 &quot;1 mL syringe&quot;)</td></tr><tr><td>BD Precisionglide syringe needles gauge 26, L 1/2 in.</td><td>Sigma-Aldrich</td><td>Z192392-100EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.21 &quot;26G, &amp;frac12;&amp;rdquo; needle&quot;)</td></tr><tr><td>Nestlets Nesting Material</td><td>Ancare</td><td>NES3600</td><td>See &quot;4. In utero electroporation&quot; (step 4.24 &quot;nesting materials&quot;)</td></tr><tr><td>Sunflower Seeds, Black Oil, Sterile</td><td>Bio-Serv</td><td>S5137</td><td>See &quot;4. In utero electroporation&quot; (step 4.24 &quot;sunflower seeds&quot;)</td></tr><tr><td>Paraformaldehyde, 97%</td><td>Alfa Aesar</td><td>A11313</td><td>See &quot;5. Histology&quot; (step 5.1.1 &quot;PFA&quot;)</td></tr><tr><td>Economy Tweezers #3, 11 cm, 0.2 x 0.4 mm tips</td><td>World Precision Instruments</td><td>#501976</td><td>See &quot;5. Histology&quot; (step 5.5 &quot;tweezers&quot;)</td></tr><tr><td>Agar powder</td><td>Alfa Aesar</td><td>#10752</td><td>See &quot;5. Histology&quot; (step 5.8.1 &quot;agar&quot;)</td></tr><tr><td>Single-edge razor blades, #9 blade</td><td>Stanley Tools</td><td>#11-515</td><td>See &quot;5. Histology&quot; (step 5.9 &quot;single-edge razor blade&quot;)</td></tr><tr><td>Specimen disc S D 50 mm</td><td>Leica</td><td>#14046327404</td><td>See &quot;5. Histology&quot; (step 5.9 &quot;vibrating microtome specimen disc&quot;)</td></tr><tr><td>Buffer tray S assembly</td><td>Leica</td><td>#1404630132</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;buffer tray&quot;)</td></tr><tr><td>VT1000 S Vibratome</td><td>Leica</td><td>#14047235612</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;vibrating microtome&quot;)</td></tr><tr><td>Double Edge Razor Blades</td><td>Personna</td><td>BP9020</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;blade&quot;)</td></tr><tr><td>Knife Holder S</td><td>Leica</td><td>#14046230131</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;knife holder&quot;)</td></tr><tr><td>Studio Elements Golden Taklon Short Handle Round Brush Set</td><td>Amazon</td><td>B0089KU6XE</td><td>See &quot;5. Histology&quot; (step 5.12.1 &quot;fine tipped paintbrush&quot;)</td></tr><tr><td>Superfrost Plus Slides</td><td>Electron Microscopy Services</td><td>#71869-11</td><td>See &quot;5. Histology&quot; (step 5.12.1 &quot;microscope slide&quot;)</td></tr><tr><td>ProLong Diamond Antifade Mountant, 10 ml</td><td>Thermofisher</td><td>P36970</td><td>See &quot;5. Histology&quot; (step 5.12.3-5.12.4 &quot;mountant&quot;)</td></tr><tr><td>Cover Glass, 24 x 50 mm, No. 1</td><td>Phenix Research Products</td><td>MS1415-10</td><td>See &quot;5. Histology&quot; (step 5.12.4 &quot;coverslip&quot;)</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td><td>Sigma-Aldrich</td><td>D9542</td><td>See &quot;5. Histology&quot; (step 5.13.1 &quot;DAPI&quot;)</td></tr><tr><td>Fixed Stage Upright Microscope</td><td>Olympus</td><td>BX51WI</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;light microscope&quot;)</td></tr><tr><td>Laser Scanning Confocal Microscope</td><td>Nikon</td><td>TE2000/C2si</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;confocal microscope&quot;)</td></tr><tr><td>4x objective, NA = 0.20</td><td>Nikon</td><td>CFI Plan Apo Lambda 4X</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;low-power objective&quot;)</td></tr><tr><td>20x objective, NA = 0.75</td><td>Nikon</td><td>CFI Plan Apo Lambda 20X</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;medium-power objective&quot;)</td></tr><tr><td>60x objective, NA = 1.40</td><td>Nikon</td><td>CFI Plan Apo VC 60X Oil</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;high-power objective&quot;)</td></tr></tbody></table>

inducing cre-lox recombination in mouse cerebral cortex through in utero electroporation

Zelle-autonome neuronale Funktionen der Gene können aufgedeckt werden, indem Sie verursachen Verlust oder Gewinn der Funktion eines Gens in einer kleinen und spärlich Population von Neuronen. Dazu muss erzeugen ein Mosaik in die Neuronen mit Verlust oder Gewinn der Funktion eines Gens von genetisch unbeirrt Gewebe umgeben sind. Hier verbinden wir das Cre-Lox-Rekombinationssystem mit in Utero Elektroporation um Mosaik Hirngewebe zu generieren, die verwendet werden, um die Zelle-autonome Funktion von Genen in Neuronen zu untersuchen. DNA-Konstrukte (erhältlich über Repositories), Codierung für eine fluoreszierende Label und Cre-Rekombinase werden in kortikalen Neuronen mit Genen flankiert mit LoxP-Sites in den Gehirnen von Mäuseembryonen in Utero mit Entwicklung eingebracht Elektroporation. Darüber hinaus beschreiben wir verschiedene Anpassungen, die in Utero Elektroporation-Methode, die Überlebensfähigkeit und Reproduzierbarkeit zu erhöhen. Diese Methode erfordert auch einen Titer für Cre-vermittelten Rekombination in einer spärlich oder Dichte Bevölkerung der Neuronen. Histologische Präparate von beschrifteten Hirngewebe nicht erforderlich (aber kann angepasst werden) Immunohistochemistry. Die Konstrukte verwendet garantieren, dass das Eindringmittel Neuronen tragen das Gen für Cre-Rekombinase beschriftet. Histologische Präparate können morphologische Analyse von Neuronen durch konfokale Bildgebung der dendritischen und axonalen Lauben und dendritischen Dornen. Weil Verlust oder Gewinn der Funktion im spärlich Mosaik Gewebe erreicht wird, erlaubt diese Methode das Studium der Zelle-autonome Notwendigkeit und Angemessenheit der Gen-Produkte in Vivo.

Die einzelnen Konstrukt GFP. CRE (siehe Materialliste) war elektroporiert bei E15.5 und visualisiert auf P14. Abhängig von der Konzentration des Konstrukts und das Volumen der Injektion erhalten Sie ein spärliches oder dichtes Ergebnis22,26. Beispiel: Injektion von 1 µL von 2 mg/mL GFP. CRE-Ergebnisse in eine spärliche Verteilung der markierten Zellen, von die einige helle (Abbildung 1A) und lokal...

Watch this Scientific Journal Video about Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation at JoVE.com

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

Zelle-autonome Funktionen der Gene im Gehirn können durch Induktion Verlust studiert werden oder der Funktion in geringer Dichte Bevölkerungen der Zellen gewinnen. Hier beschreiben wir in Utero Elektroporation Cre-Rekombinase in spärlichen Bevölkerung kortikale Neuronen mit Floxed Genen zu entwickeln, Verlust der Funktion in Vivozu liefern.

Induktion Cre Lox Rekombination in Maus Großhirnrinde durch Elektroporation In Utero

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 ...

inducing-cre-lox-recombination-mouse-cerebral-cortex-through-utero

Research

JoVE Journal

Neuroscience

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

11.6K Aufrufe.  James Madison University. Zelle-autonome Funktionen der Gene im Gehirn können durch Induktion Verlust studiert werden oder der Funktion in geringer Dichte Bevölkerungen der Zellen gewinnen. Hier beschreiben wir in Utero Elektroporation Cre-Rekombinase in spärlichen Bevölkerung kortikale Neuronen mit Floxed Genen zu entwickeln, Verlust der Funktion in Vivozu liefern.

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

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

Induktion der Protein Deletion Durch<em&gt; In Utero</em&gt; Elektroporation, um Defizite in Neuronale Migration in Transgenic Models definieren

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

Forschung

Entwicklungsbiologie

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In Utero intraventrikuläre Injektion und Elektroporation von E15 muriner Embryonen

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Biologie

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.

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 ...

Neurowissenschaften

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.

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.

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 ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

Methoden zur Untersuchung der neuronalen Morphogenese:<em&gt; Ex-vivo-</em&gt; RNAi Elektroporation in embryonale Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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.

Visualisierung und genetische Manipulation von Dendriten und Dornen in der Maus Hirnrinde und im Hippocampus mit<em&gt; In utero</em&gt; Die Elektroporation

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 ...

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.

Cortex, Hippocampus-, Thalamus-, Hypothalamus, Lateral Septal Nucleus- und Striatum spezifische<em&gt; In Utero</em&gt; Electroporation in der C57BL / 6-Maus,

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

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, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

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

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

In Utero Ansätze Elektroporation die Erregbarkeit neuronaler Subpopulationen und Einzelzell - Konnektivität zu studieren

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

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

Double In Utero Elektroporation, um zeitlich und räumlich getrennte Zellpopulationen anzuvisieren

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

In Utero Elektroporation von Multiadressierbaren Genom-integrierenden Farbe (MAGIC) Marker zur Individualisierung kortikaler Maus-Astrozyten

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

Induktion Cre Lox Rekombination in Maus Großhirnrinde durch Elektroporation In Utero

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Methoden zur Untersuchung der neuronalen Morphogenese:

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