Name: stereotaxic surgery for genetic manipulation in striatal cells of neonatal mouse brains
Uploaded: 2018-07-10T12:30:00
Description: Watch this Scientific Journal Video about Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains at JoVE.com

Dette arbejde blev støttet af Ministeriet for videnskab og teknologi giver MOST104-2311-B-010-010-MY3, MOST106-2321-B-010-012, de nationale sundheds-forskningsinstitutter give NHRI-EX106-10429NI, og de fremhævede områder Research Center Program tilskud fra Undervisningsministeriet gennem Brain Forskningscenter, nationale Yang-Ming universitetet i Taiwan, og postdoc stipendium giver MOST106-2811-B-010-031 (S.-YC), MOST105-2811-B-010-036 og MOST106-2811-B-010-030 (H.-Y.K.).

De animalske protokoller er beskrevet her er blevet godkendt af Animal Care og brug udvalgene af nationale Yang-Ming Universitet.
1. forberedelse af indehaveren til Neonatal unger i den stereotaxisk apparatur
<ol><li>Gøre bakken hoved: skær bunden af en 1,5 mL centrifugeglas (15 mm lange) i den form, der passer til overhoved neonatale unger ved at fjerne 1/5 af mur af røret.</li>
	<li>Tag en pipette tip box med den rigtige størrelse, der passer med piedestalen af stereotaxisk apparatur, ...

<ol>
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(77), e50600 (2013).</li><li>Mathon, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+effectiveness+of+intracerebral+injections+in+adult+and+neonatal+mice:+a+neurosurgical+point+of+view.">Increasing the effectiveness of intracerebral injections in adult and neonatal mice: a neurosurgical point of view.</a> Neurosci. Bull. 31 (6), 685-696 (2015).</li><li>Davidson, S., Truong, H., Nakagawa, Y., Giesler, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microinjection+technique+for+targeting+regions+of+embryonic+and+neonatal+mouse+brain+in+vivo.">A microinjection technique for targeting regions of embryonic and neonatal mouse brain in vivo.</a> Brain Res. 1307, 43-52 (2010).</li><li>Chen, Y. 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I den foreliggende undersøgelse viser vi en enkel og pålidelig stereotaxisk metode til indsprøjtning AAV vira i striatum af neonatal mus hjerner. Vi microinjected AAV-eGFP-Cre vira i striatum af Ai14 reporter mus på P2 og derefter analyseret reporter gen expression på P14. Vi fandt AAV transduced normal god landbrugspraksis-positive celler i hele striatum på rostrocaudal niveauer. Desuden, næsten alle normal god landbrugspraksis-positive celler Co udtrykt tdTomato reporter gen i striatal celler, hvilket tyder på ...

Moderne studier af struktur og funktion af hjernen kræver normalt genmanipulation af specifikke gener i neuronale celler. For at sonde funktioner af forskellige gener, Transgene mus transporterer mutante alleler, herunder er knockout og knock-i alleler rutinemæssigt genereret. Stereotaxisk hjerneoperation for voksen gnavere er en standardmetode til lokalt levere narkotika, vira, sporstoffer og andre reagenser til bestemte områder af gnaver hjerner1,2. Anvendelse af stereotaxisk hjerneoperation Transgene mus tillader en at genmanipulere genfunktioner og neuronal aktivitet i specifikke neuronal populationer af musen hjernen. Celle type-specifikke manipulation giver en kraftfuld tilgang for at dechifrere neuronal funktioner i komplekse neurale kredsløb i hjernen3,4,5.
Neurale udvikling af nervesystemet begynder på tidlige vorden, og de udviklingsmæssige processer fortsætter efter fødslen indtil den juvenile periode. Postnatal modning af nervesystemet omfatter den præcise synaptic ledninger af neurale kredsløb, der er afgørende for fysiologiske og kognitive funktioner i hjernen6. Derfor, at studere udviklingsmæssige begivenheder, der opstår i neonatal tiden vinduer er vigtigt ikke kun for at forstå normal neurale udvikling, men det kan også give indsigt i patogenesen af udviklingsforstyrrelser og neuropsykiatriske lidelser7 ,8. Selv om metoder af stereotaxisk hjerneoperation for voksen gnavere er let tilgængelige2,9, er par protokoller tilgængelige på internettet for stereotaxisk hjerneoperation i neonatal mus10,11. Faktisk er stereotaxisk microinjections af reagenser i hjerner af neonatal mus unger vanskeligt, fordi overhoved neonatale pup er for skrøbelige til at blive fast i den standard stereotaxisk apparatur. Ikke desto mindre er anvendelsen af stereotaxisk hjerneoperation på Transgene mus muligt for neonatal mus12. Her, beskriver vi en enkel metode med en hjemmelavet setup til at udføre stereotaxisk hjerneoperation i nyfødte mus unger. Vi demonstrere, at denne teknik giver mulighed for en betinget slette floxed gener af microinjecting AAV-udtrykker Cre DNA recombinase i striatum reporter gen mus og betinget floxed Transgene mus. Denne teknik gælder også levere reagenser i wild-type mus neonatal striatum.

<table border="0" cellpadding="0" cellspacing="0" width="1134">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">30G PrecisionGlide Needle</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">REF 305106</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Chloroform</td>
			<td style="width:273px;">JT Baker</td>
			<td style="width:199px;">9180-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Hamilton MICROLITER Syringe</td>
			<td style="width:273px;">Hamilton&#160;</td>
			<td style="width:199px;">80300</td>
			<td style="width:395px;">30G needle fit for PE10 tube; 26G needle needs a PE20 adaptor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Polyethylene tubing PE20</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">427406</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Polyethylene tubing PE10</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">427401</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Micro Flow Rate Syringe Pump</td>
			<td style="width:273px;">Longer Precision Pump Co.</td>
			<td style="width:199px;">TJ-2A (Controller) and L0107-2A (Drive Unit)</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">25G syringe</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">REF 302105</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Fast green</td>
			<td style="width:273px;">Sigma-Aldrich</td>
			<td style="width:199px;">F-7252</td>
			<td style="width:395px;">0.1%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Standard Stereotaxic Instruments</td>
			<td style="width:273px;">RWD Life Science</td>
			<td style="width:199px;">68037</td>
			<td style="width:395px;">Without using 68030 Mouse/Neonatal Rat Adaptor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Anti-FOXP2 antibody</td>
			<td style="width:273px;">Abcam</td>
			<td style="width:199px;">ab16046</td>
			<td style="width:395px;">Rabbit polyclonal to FOXP2, 1:4K</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Anti-RFP antibody</td>
			<td style="width:273px;">Abcam</td>
			<td style="width:199px;">ab65856</td>
			<td style="width:395px;">Mouse monoclonal to RFP, 1:1K</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">BX63 microscope</td>
			<td style="width:273px;">Olympus</td>
			<td style="width:199px;">BX63</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">LSM 880 confocal microscope</td>
			<td style="width:273px;">Zeiss</td>
			<td style="width:199px;">LSM 880</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Goat anti-rabbit conjugated Alexa fluor594</td>
			<td style="width:273px;">Jackson lmmunoReserch Laboratories Inc.</td>
			<td style="width:199px;">111-585-003</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40</td>
			<td style="width:273px;">Penn Vector Core</td>
			<td style="width:199px;">AV-9-PV1848</td>
			<td style="width:395px;">Lot # CS0987, 5.506x1013 (GC/mL)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">AAV9.chicken actin-eGFP</td>
			<td style="width:273px;">AAV core, Institute of Biomedical Sciences, Academia Sinica, Taiwan</td>
			<td style="width:199px;">N/A</td>
			<td style="width:395px;">1x1014 (GC/ml)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:59px;width:269px;">B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td>
			<td style="width:273px;">The Jackson Labtorary&#160;</td>
			<td style="width:199px;">007914</td>
			<td>Ai14</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">B6(Cg)-Foxp2tm1.1Sfis/CfreJ</td>
			<td style="width:273px;">The Jackson Labtorary&#160;</td>
			<td style="width:199px;">026259</td>
			<td style="width:395px;">Foxp2fl/fl</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Dulbecco&#8217;s phosphate buffered saline</td>
			<td style="width:273px;">Corning&#160;cellgro</td>
			<td style="width:199px;">21-030-CVR</td>
			<td></td>
		</tr>
	</tbody>
</table>

stereotaxic surgery for genetic manipulation in striatal cells of neonatal mouse brains

Mange gener er udtrykt i embryonale hjerner, og nogle af dem er løbende udtrykt i hjernen efter fødslen. For sådanne vedvarende udtrykte gener, kan de fungere for at regulere den udviklingsmæssige proces og/eller fysiologiske funktion i neonatal hjerner. For at undersøge neurobiologiske funktioner af specifikke gener i hjernen, er det vigtigt at inaktivere gener i hjernen. Her, beskriver vi en simpel stereotaxisk metode til at inaktivere genekspression i striatum af Transgene mus på neonatal tidsvinduer. AAV-eGFP-Cre virus var microinjected i striatum af Ai14 reporter gen mus på postnatal dag (P) 2 af stereotaxisk hjernekirurgi. TdTomato reporter gen expression blev opdaget i P14 striatum, hvilket tyder på en vellykket Cre-loxP medieret DNA rekombination i AAV-transduced striatal celler. Vi yderligere valideret denne teknik ved microinjecting AAV-eGFP-Cre vira til P2Foxp2fl/fl mus. Dobbelt mærkning af normal god landbrugspraksis og Foxp2 viste, at normal god landbrugspraksis-positive celler manglede Foxp2 immunoreactivity i P9 striatum, hvilket tyder på tabet af Foxp2 protein i AAV-eGFP-Cre transduced striatal celler. Taget sammen, viser disse resultater en effektiv genetiske sletning af stereotaxically microinjected AAV-eGFP-Cre vira i specifikke neuronal populationer i neonatal hjerner af floxed Transgene mus. Afslutningsvis, giver vores stereotaxisk teknik en nem og simpel platform for genmanipulation i neonatal mus hjerner. Teknikken kan ikke kun bruges til at slette gener i specifikke regioner af neonatal hjerner, men det kan også bruges til at injicere farmakologiske stoffer, neuronal røbestoffer, genetisk modificerede optogenetics og chemogenetics proteiner, neuronal aktivitet indikatorer og andre reagenser i striatum af neonatal mus hjerner.

For det første eksperiment, vi microinjected 200 nL af AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40 vira (AAV-eGFP-Cre, 1/10 fortynding i Dulbeccos fosfatbufferet saltopløsning), der udtrykker Cre DNA recombinase sammenvokset med normal god landbrugspraksis i P2 striatum af Ai14 mus. Ai14 mus express tdTomato reporter gen ved Cre-medieret sletning af loxP-flankeret (floxed) STOP kassette (figur 2F). Hjerner er høstet på P14 for immunfarvning af normal god landbru...

Watch this Scientific Journal Video about Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains at JoVE.com

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Stereotaxisk kirurgi for genmanipulation i Striatal celler af Neonatal mus hjerner

Vi beskriver en protokol af stereotaxisk kirurgi med en hjemmelavet hoved-fast enhed for microinjecting reagenser i striatum af neonatal mus hjerner. Denne teknik gør det muligt for genmanipulation neuronale celler i bestemte regioner af neonatal mus hjerner.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

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