Name: inducing cre-lox recombination in mouse cerebral cortex through in utero electroporation
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Forfatterne takker sjenerøs støtte fra James Madison University Institutt for biologi og James Madison University lys mikroskopi og Imaging anlegg. Dr. Mark L. Gabriele for nyttige råd om unge postnatal vev forberedelse, og Dr. Justin W. Brown og Corey L. Cleland for sjenerøs koordinering av kirurgiske materialer og plass. Denne forskningen ble finansiert delvis av et samarbeidsprosjekt forskningsstipend 4-VA, et samarbeidsprosjekt samarbeid for fremme Samveldet av Virginia (G.S.V.) og av et Virginia akademiet av vitenskap liten prosjektet forskningsstipend (G.S.V.). Støtte har blitt sjenerøst gitt av en Betty Jo kjærlig Butler 58 begavelse for Undergraduate Research stipend (til K.M.B.), en Farrell sommer forskning stipend (til K.M.B.), James Madison University andre århundre stipend (til K.M.B.), en James Madison University Centennial stipend (til C.J.H.), James Madison University Lucy Robinson søk ' 30 Memorial stipend (til Z.L.H.) og en James Madison University College vitenskap og matematikk fakultetet Assistance Grant (til G.S.V.).

Metodene som er beskrevet her er godkjent av Animal Care og bruk Committee (ACUC) ved James Madison University, og er i samsvar og overholdelse av alle relevante regulatoriske og institusjonelle retningslinjer.
1. musen oppsett
<ol><li>Huset en ung (> P60) mannlige og kvinnelige homozygous floxed mus sammen for å sette opp en oppdretter par25. Merk: En god negativ kontroll er å sette opp en ekstra oppdretter par wildtype mus, i hvilken grobunn recombinase uttrykk...

<ol>
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Her introduserer vi kombinasjonen av i utero electroporation med grobunn recombinase i floxed mus å generere mosaikk hjernevev. En fordel med denne tilnærmingen er at en ny mus linje ikke trenger genereres hver gang en annen mobilnettet undertype skal målrettes: i utero electroporation kan brukes til å målrette eksitatoriske nerveceller, hemmende nerveceller eller glia avhengig og plassering av electroporation15,16,<sup class="xr...

Generere en genetisk mosaikk er et klassisk eksperimentelle paradigme for å forstå funksjonen av et genet av interesse. For å avgjøre hvis en genet er nødvendig for en mobilnettet fenotype, forårsaker den enkleste måten tap av funksjon av genet hele organismen (f.eks knockout). Men for å avgjøre om et gen er nødvendig i en bestemt celle type, er knockout av genet hele organismen ikke en gyldig tilnærming. I stedet, en metode er nødvendig som vil føre til tap av funksjon av et gen i en gitt celle mens det er omgitt av wildtype (i.e. genetisk Uaffisert) vev-med andre ord, lage mosaikk vev. Hvis mutant cellen viser en mutant fenotype, men omkringliggende wildtype cellene ikke, gene funksjonene i en celle autonome måte. Analyse av mosaikk vev, der mutant celler er omgitt av wildtype vev, er ideelt for å forstå celle autonome funksjoner av gener, spesielt i hjernen hvor neurons og glia danner en sammenhengende nettverk av vev.
Flere former for mosaikk hjernevev har gitt kraftige modeller for å undersøke celle autonome funksjoner av gener. Studier fokuserte på neuronal transplantasjon1, kvinnelige X-koblede mosaicism2,3,4, og endogene somatisk mosaicism5,6 har trukket sine konklusjoner basert på mosaic hjernevev. Betinget sletting av et gen gjennom grobunn-lox rekombinasjon systemet er en metode som drar full nytte av store tilgjengeligheten av transgene musen linjer. I denne metoden får er to loxP nettsteder innført på hver side av en obligatoriske rekkefølgen til et gen (for eksempel en ekson), la det flankert av loxP at både ansikt i samme retning ("floxed"). Grobunn recombinase avgifter rekkefølgen mellom loxP nettsteder7. Grobunn-mediert rekombinasjon kan oppnås ved krysset floxed mus til en annen mus linje uttrykke grobunn recombinase sammen med merketråd fluorescerende i en gruppe celler ("grobunn reporter linje"). Dette har blitt vist på en rekke måter å avdekke funksjonene til et gen i undergrupper av celler, for eksempel eksitatoriske nerveceller eller astrocyttene8. Grobunn reporter linjer kan uttrykke CreERT2 å tillate grobunn-mediert rekombinasjon skal narkotika-induserbart (single-Nevron merking med induserbart grobunn-mediert knockout eller GLATT)9. En annen strategi kalt mosaikk analyse med dobbel markører (MADM)10,11, kan grobunn-mediert interchromosomal rekombinasjon en homozygous mutant opprettes sammen med heterozygote vev. Disse tilnærmingene må en ny linje av mus produseres hver gang for hver kandidat genet eller cellular undertype som testes. Alternativt kan grobunn recombinase innføres fødselen via Iontoforese12 eller viral vektorer (f.eks adeno-assosiert virus13 eller lentiviruses14 bærer mobilnettet Undertypespesifikke Arrangørene). Denne strategien skaper sterke og postnatal merking. Målrettingsland utvikle cerebral kortikale nevroner tynt og prenatally er en perfekt strategi i utero electroporation av grobunn recombinase med merketråd fluorescerende.
I tillegg kombinerer grobunn-lox rekombinasjon gjennom i utero electroporation å produsere mosaikk vev i vivo, vi introdusere flere tilpasninger til prosedyrer fra andre publiserte protokoller15,16, 17,18,19,20,21. Vi gir informasjon for å forbedre suksessen i avl tidsbestemte-gravide kvinner. Vi også skissere våre to strategier for å innføre sparsom og lyse merking av nerveceller i kortikale vev: en strategi er å sjarmere nivåer av en enkelt konstruere koding for grobunn recombinase og en fluorescerende markør22. En annen strategi er å bruke "Supernova" systemet, designet spesielt med disse parameterne i tankene23,24. I tillegg tilbyr vi forbedringer på konsekvent microinjection Pipetter og forenklinger til i utero electroporation kirurgi. Til slutt, vi skissere avgjørende skritt i en forenklet histologiske forberedelse som tillater analyse av dendrittiske spines og dendrittiske og axonal arbors, uten ytterligere flekker eller immunohistochemistry.

<table>
	<tbody>
		<tr>
			<td>C57BL/6J mice</td>
			<td>The Jackson Laboratory</td>
			<td>#000664</td>
			<td>See &#34;1. Mouse set-up&#34; (step 1.1, &#34;wildtype mice&#34;)</td>
		</tr>
		<tr>
			<td>GFP.Cre empty vector</td>
			<td>AddGene</td>
			<td>#20781</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.1 &#34;single DNA construct that codes for Cre recombinase as well as a fluorescent marker&#34;). 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 &#34;2. DNA set-up&#34; (step 2.1 &#34;Supernova&#34; 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 &#34;2. DNA set-up&#34; (step 2.1 &#34;Supernova&#34; 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 &#34;2. DNA set-up&#34; (step 2.1 &#34;Supernova&#34; 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 &#34;2. DNA set-up&#34; (step 2.3 &#34;endotoxin-free plasmid purification kit&#34;)</td>
		</tr>
		<tr>
			<td>Trypan Blue powder, BioReagent grade</td>
			<td>Sigma</td>
			<td>T6146-5G</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.5 &#34;trypan blue&#34;)</td>
		</tr>
		<tr>
			<td>Sodium Chloride, ACS, 2.5 kg</td>
			<td>VWR</td>
			<td>BDH9286-2.5KG</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.5 &#34;NaCl&#34;)</td>
		</tr>
		<tr>
			<td>Potassium Chloride, ACS, 500 g</td>
			<td>VWR</td>
			<td>#97061-566</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.5 &#34;KCl&#34;)</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic, ReagentPlus, 100 g</td>
			<td>Sigma-Aldrich</td>
			<td>S0876-100G</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.5 &#34;Na2HPO4&#34;)</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic, ReagentPlus, 100 g</td>
			<td>Sigma-Aldrich</td>
			<td>P5379-100G</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.5 &#34;KH2PO4&#34;)</td>
		</tr>
		<tr>
			<td>Hydrochloric acid, ACS reagent, 500 mL</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>See &#34;2. DNA set-up&#34; (step 2.5 &#34;HCl&#34;)</td>
		</tr>
		<tr>
			<td>P-97 Micropipette Puller</td>
			<td>Sutter Instrument</td>
			<td>P-97</td>
			<td>See &#34;3. Pipette set-up&#34; (step 3.1 &#34;glass capillary puller&#34;)</td>
		</tr>
		<tr>
			<td>3.0 mm wide trough filament</td>
			<td>Sutter Instrument</td>
			<td>FT330B</td>
			<td>See &#34;3. Pipette set-up&#34; (step 3.1 &#34;glass capillary puller&#34;)</td>
		</tr>
		<tr>
			<td>Thin Wall Glass Capillaries, 4&#34;, 1 / 0.75 OD/ID</td>
			<td>World Precision Instruments</td>
			<td>TW100-4</td>
			<td>See &#34;3. Pipette set-up&#34; (step 3.1.1 &#34;glass capillary&#34;)</td>
		</tr>
		<tr>
			<td>Single Ply Soft-Tech Wipes, 4.5&#34;</td>
			<td>Phenix</td>
			<td>LW-8148</td>
			<td>See &#34;3. Pipette set-up&#34; (step 3.2.1 &#34;single-ply task wipe&#34;); 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 &#34;4. In utero electroporation&#34; (step 4.1 &#34;Graefe forceps&#34;)</td>
		</tr>
		<tr>
			<td>Iris Scissors, 11.5 cm, Straight, 12-pack</td>
			<td>World Precision Instruments</td>
			<td>#503708-12</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;iris scissors&#34;)</td>
		</tr>
		<tr>
			<td>Hartman Mosquito Forceps, 9 cm, Straight, 12-pack</td>
			<td>World Precision Instruments</td>
			<td>#503728-12</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;Hartman mosquito forceps&#34;)</td>
		</tr>
		<tr>
			<td>General Purpose Non-Woven Sponges, 2&#34; x 2&#34;, 4-ply</td>
			<td>Medrepexpress</td>
			<td>#2204-c</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;non-woven gauze sponges&#34;)</td>
		</tr>
		<tr>
			<td>Ring Tipped Forceps, 10 cm, Straight, 2.2mm ID</td>
			<td>World Precision Instruments</td>
			<td>#503203</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;ring-tipped forceps&#34;)</td>
		</tr>
		<tr>
			<td>Pyrex petri dishes complete, O.D. &#215; H 100 mm &#215; 20 mm</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3160102-12EA</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;Petri dishes&#34;)</td>
		</tr>
		<tr>
			<td>Flat Type Instrument Tray, Stainless Steel, 13-5/8&#34; x 9-3/4&#34; x 5/8&#34;</td>
			<td>Amazon</td>
			<td>B007SHGAHA</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.1 &#34;stainless steel tray&#34;)</td>
		</tr>
		<tr>
			<td>Platinum Tweezertrode, 5 mm</td>
			<td>BTX</td>
			<td>#45-0489</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.3 and 4.16 &#34;tweezer-type electrodes&#34;)</td>
		</tr>
		<tr>
			<td>ECM 830 Foot Pedal</td>
			<td>BTX</td>
			<td>#45-0211</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.3 and 4.17 &#34;foot pedal&#34;)</td>
		</tr>
		<tr>
			<td>ECM 830 Generator</td>
			<td>BTX</td>
			<td>#45-0052</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.3 &#34;generator&#34;)</td>
		</tr>
		<tr>
			<td>Single Animal Isoflurane Anesthesia System with Small Induction Box</td>
			<td>Harvard Apparatus</td>
			<td>#72-6468</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.4 and 4.6 &#34;nose cone&#34;, step 4.4 &#34;induction chamber&#34;)</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Hanna Pharmaceutical Supply Co</td>
			<td>#0536108691</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.7 &#34;veterinary ophthalmic ointment&#34;)</td>
		</tr>
		<tr>
			<td>Space Gel (AIMS)</td>
			<td>VWR</td>
			<td>#95059-640</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.8 &#34;sealed pouch filled with supersaturated salt solution&#34;)</td>
		</tr>
		<tr>
			<td>Hair Remover Gel Cream, Sensitive Formula</td>
			<td>Veet</td>
			<td>#062200809951</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.9 &#34;depilatory cream&#34;)</td>
		</tr>
		<tr>
			<td>10ul Low Retention Tip Starter (960 tips/pk)</td>
			<td>Phenix Research Products</td>
			<td>TSP-10LKIT</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.12 &#34;sterile 10 &#181;L micropipette tip&#34;)</td>
		</tr>
		<tr>
			<td>Aspirator tube assemblies for calibrated microcapillary pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>A5177</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.15 &#34;aspirator tube assembly&#34;)</td>
		</tr>
		<tr>
			<td>Braided Absorbable Suture, 4-0, Needle NFS-2(FS-2), 27&#34;</td>
			<td>Medrepexpress</td>
			<td>MV-J397</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.19 &#34;absorbable sutures&#34;)</td>
		</tr>
		<tr>
			<td>&#8220;LiquiVet Rapid&#8221; Tissue Adhesive</td>
			<td>Medrepexpress</td>
			<td>VG3</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.20 &#34;tissue adhesive&#34;)</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 &#34;4. In utero electroporation&#34; (step 4.21 &#34;1 mL syringe&#34;)</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 &#34;4. In utero electroporation&#34; (step 4.21 &#34;26G, &#189;&#8221; needle&#34;)</td>
		</tr>
		<tr>
			<td>Nestlets Nesting Material</td>
			<td>Ancare</td>
			<td>NES3600</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.24 &#34;nesting materials&#34;)</td>
		</tr>
		<tr>
			<td>Sunflower Seeds, Black Oil, Sterile</td>
			<td>Bio-Serv</td>
			<td>S5137</td>
			<td>See &#34;4. In utero electroporation&#34; (step 4.24 &#34;sunflower seeds&#34;)</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 97%</td>
			<td>Alfa Aesar</td>
			<td>A11313</td>
			<td>See &#34;5. Histology&#34; (step 5.1.1 &#34;PFA&#34;)</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 &#34;5. Histology&#34; (step 5.5 &#34;tweezers&#34;)</td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>Alfa Aesar</td>
			<td>#10752</td>
			<td>See &#34;5. Histology&#34; (step 5.8.1 &#34;agar&#34;)</td>
		</tr>
		<tr>
			<td>Single-edge razor blades, #9 blade</td>
			<td>Stanley Tools</td>
			<td>#11-515</td>
			<td>See &#34;5. Histology&#34; (step 5.9 &#34;single-edge razor blade&#34;)</td>
		</tr>
		<tr>
			<td>Specimen disc S D 50 mm</td>
			<td>Leica</td>
			<td>#14046327404</td>
			<td>See &#34;5. Histology&#34; (step 5.9 &#34;vibrating microtome specimen disc&#34;)</td>
		</tr>
		<tr>
			<td>Buffer tray S assembly</td>
			<td>Leica</td>
			<td>#1404630132</td>
			<td>See &#34;5. Histology&#34; (step 5.10 &#34;buffer tray&#34;)</td>
		</tr>
		<tr>
			<td>VT1000 S Vibratome</td>
			<td>Leica</td>
			<td>#14047235612</td>
			<td>See &#34;5. Histology&#34; (step 5.10 &#34;vibrating microtome&#34;)</td>
		</tr>
		<tr>
			<td>Double Edge Razor Blades</td>
			<td>Personna</td>
			<td>BP9020</td>
			<td>See &#34;5. Histology&#34; (step 5.10 &#34;blade&#34;)</td>
		</tr>
		<tr>
			<td>Knife Holder S</td>
			<td>Leica</td>
			<td>#14046230131</td>
			<td>See &#34;5. Histology&#34; (step 5.10 &#34;knife holder&#34;)</td>
		</tr>
		<tr>
			<td>Studio Elements Golden Taklon Short Handle Round Brush Set</td>
			<td>Amazon</td>
			<td>B0089KU6XE</td>
			<td>See &#34;5. Histology&#34; (step 5.12.1 &#34;fine tipped paintbrush&#34;)</td>
		</tr>
		<tr>
			<td>Superfrost Plus Slides</td>
			<td>Electron Microscopy Services</td>
			<td>#71869-11</td>
			<td>See &#34;5. Histology&#34; (step 5.12.1 &#34;microscope slide&#34;)</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant, 10 ml</td>
			<td>Thermofisher</td>
			<td>P36970</td>
			<td>See &#34;5. Histology&#34; (step 5.12.3-5.12.4 &#34;mountant&#34;)</td>
		</tr>
		<tr>
			<td>Cover Glass, 24 x 50 mm, No. 1</td>
			<td>Phenix Research Products</td>
			<td>MS1415-10</td>
			<td>See &#34;5. Histology&#34; (step 5.12.4 &#34;coverslip&#34;)</td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>See &#34;5. Histology&#34; (step 5.13.1 &#34;DAPI&#34;)</td>
		</tr>
		<tr>
			<td>Fixed Stage Upright Microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>See &#34;5. Histology&#34; (step 5.15 &#34;light microscope&#34;)</td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>Nikon</td>
			<td>TE2000/C2si</td>
			<td>See &#34;5. Histology&#34; (step 5.15 &#34;confocal microscope&#34;)</td>
		</tr>
		<tr>
			<td>4x objective, NA = 0.20</td>
			<td>Nikon</td>
			<td>CFI Plan Apo Lambda 4X</td>
			<td>See &#34;5. Histology&#34; (step 5.15 &#34;low-power objective&#34;)</td>
		</tr>
		<tr>
			<td>20x objective, NA = 0.75</td>
			<td>Nikon</td>
			<td>CFI Plan Apo Lambda 20X</td>
			<td>See &#34;5. Histology&#34; (step 5.15 &#34;medium-power objective&#34;)</td>
		</tr>
		<tr>
			<td>60x objective, NA = 1.40</td>
			<td>Nikon</td>
			<td>CFI Plan Apo VC 60X Oil</td>
			<td>See &#34;5. Histology&#34; (step 5.15 &#34;high-power objective&#34;)</td>
		</tr>
	</tbody>
</table>

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

Cellen autonome neuronal funksjoner av gener kan vises ved forårsaker tap eller gevinst på funksjon av et gen i en liten og sparsom befolkning av neurons. Det krever genererer en mosaikk der neurons med tap eller fortjeneste av et gen er omgitt av genetisk Uaffisert vev. Her kombineres grobunn-lox rekombinasjon systemet med i utero electroporation for å generere mosaikk hjernevev som kan brukes til å studere funksjonen celle autonome av gener i neurons. DNA konstruksjoner (tilgjengelig i repositories), koding for et lysstoffrør etikett og grobunn recombinase, blir introdusert inn i utvikling av kortikale nevroner som inneholder gener flankert med loxP områder i hjernen til mouse embryoer bruker i utero electroporation. I tillegg beskriver vi ulike tilpasninger for metoden i utero electroporation som øker overlevelsesevne og reproduserbarhet. Denne metoden innebærer også å etablere en titer for grobunn-mediert rekombinasjon i en sparsom eller tett befolkning av neurons. Histologiske preparater av merket hjernevev ikke krever (men kan tilpasses) immunohistochemistry. Konstruksjoner brukes garanterer at fluorescently merket neurons bærer genet for grobunn recombinase. Histologiske forberedelser tillate morfologisk analyse av neurons gjennom AC confocal avbildning av dendrittiske og axonal arbors og dendrittiske spines. Fordi tap eller vinning-funksjonen er oppnådd i sparsom mosaikk vev, tillater denne metoden studiet av cellen autonome nødvendighet og tilstrekkelighet av genet produkter i vivo.

Singelen konstruere GFP. Grobunn (se liste over materialer) var electroporated på E15.5 og visualisert på P14. Avhengig av konsentrasjonen av Konstruer og volumet av injeksjon, kan et sparsommelig eller tett resultat oppnådd22,26. For eksempel injeksjon av 1 µL av 2 mg/mL GFP. Grobunn resultater i en sparsom distribusjon av merket celler, noe som kan være lys (figur 1A), og lokalisert i layer II/...

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

Cellen autonome funksjoner av gener i hjernen kan studeres ved å fremkalle tap eller gevinst på funksjonen sparsom bestander av celler. Her beskriver vi i utero electroporation gir grobunn recombinase i sparsom bestander av utvikle kortikale neurons med floxed gener forårsake tap av funksjon i vivo.

Inducing grobunn-lox rekombinasjon i musen hjernebarken gjennom In Utero Electroporation

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

Research

JoVE Journal

Neuroscience

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

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 ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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 ...

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 ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

Påvisning av Microregional Hypoksi i Mus Cerebral Cortex av to-foton Imaging av endogen NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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

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

Metoder for å studere nevrale morphogenesis:<em&gt; Ex vivo</em&gt; RNAi electroporation i Embryonic murin 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

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

Visualisering og genetisk manipulering av dendritter og pigger i Mus hjernebarken og hippocampus hjelp<em&gt; I fosterlivet</em&gt; 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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Dual Elektrofysiologiske Opptak av Synaptically-fremkalt Astroglial og Neuronal Responses i akutt hippocampus Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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

Generering av Topically Transgene Rats etter<em&gt; I utero</em&gt; Electroporation og<em&gt; In vivo</em&gt; Bioluminesens 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

Genetisk manipulering av musen Utvikling Hypothalamus gjennom<em&gt; I utero</em&gt; Electroporation

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

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

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

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- og striatum spesifikke<em&gt; I Utero</em&gt; Electroporation i C57BL / 6 mus

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