Name: methods and tips for intravenous administration of adeno-associated virus to rats and evaluation of central nervous system transduction
Uploaded: 2017-08-25T13:00:00
Description: Watch this Scientific Journal Video about Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction at JoVE.com

Dette arbejde blev finansieret af ALS Association, Karyopharm Therapeutics, Inc., Meira GTx og en velgørende donation til ALS research fra Thomas Lawson, hvilket vi er taknemmelige. Vi takker Elysse Orchard og Donna Burney for rådgivning og uddannelse. JAS blev støttet af løj Foundation og National Institutes of Health giver GM103418.

alle procedurer fulgt retningslinjerne for passende NIH. Alle procedurerne fulgt animalske protokoller, der blev godkendt ved Animal Care og brug Udvalget på LSU Health Sciences Center i Shreveport. Sprague-Dawley hunrotter henne ved 6 ugens i alder blev brugt til denne procedure.
 1. afgøre doser/mængder af vektor behov for <ol><li>vejer rotter til at blive injiceret og stoerrelsen af viral vektor behov for hvert enkelt dyr baseret på titer (fusion) af vektor forberedelse. Brug vektor ...

<ol>
	<li>Foust, K., Nurre, E., Montgomery, C., Hernandez, A., Chan, C., Kaspar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat. Biotechnol. 27 (1), 59-65 (2009).</li><li>Duque, S., 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=Intravenous+administration+of+self-complementary+AAV9+enables+transgene+delivery+to+adult+motor+neurons.">Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.</a> Mol. Ther. 17 (7), 1187-1196 (2009).</li><li>Gombash Lampe, S., Kaspar, B., Foust, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+injections+in+neonatal+mice.">Intravenous injections in neonatal mice.</a> J. Vis. Exp. (93), e52037 (2014).</li><li>Wang, D., 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=Expansive+gene+transfer+in+the+rat+CNS+rapidly+produces+amyotrophic+lateral+sclerosis+relevant+sequelae+when+TDP-43+is+overexpressed.">Expansive gene transfer in the rat CNS rapidly produces amyotrophic lateral sclerosis relevant sequelae when TDP-43 is overexpressed.</a> Mol. Ther. 18 (12), 2064-2074 (2010).</li><li>Dayton, R., 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=Selective+forelimb+impairment+in+rats+expressing+a+pathological+TDP-43+25+kDa+C-terminal+fragment+to+mimic+amyotrophic+lateral+sclerosis.">Selective forelimb impairment in rats expressing a pathological TDP-43 25 kDa C-terminal fragment to mimic amyotrophic lateral sclerosis.</a> Mol. Ther. 21 (7), 1324-1334 (2013).</li><li>Jackson, K., Dayton, R., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV9+supports+wide-scale+transduction+of+the+CNS+and+TDP-43+disease+modeling+in+adult+rats.">AAV9 supports wide-scale transduction of the CNS and TDP-43 disease modeling in adult rats.</a> Mol. Ther. Methods Clin. Dev. 2, 15036 (2015).</li><li>Jackson, K., 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=Preservation+of+forelimb+function+by+UPF1+gene+therapy+in+a+rat+model+of+TDP-43-induced+paralysis.">Preservation of forelimb function by UPF1 gene therapy in a rat model of TDP-43-induced paralysis.</a> Gene Therapy. 22 (1), 20-28 (2015).</li><li>Jackson, K., Dayton, R., Deverman, B., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Better+targeting,+better+efficiency+for+wide-scale+neuronal+transduction+with+the+synapsin+promoter+and+AAV-PHP.B.">Better targeting, better efficiency for wide-scale neuronal transduction with the synapsin promoter and AAV-PHP.B.</a> Front. Mol. Neurosci. 9, 116 (2016).</li><li>Jackson, K., 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=Severe+respiratory+changes+at+end+stage+in+a+FUS-induced+disease+state+in+adult+rats.">Severe respiratory changes at end stage in a FUS-induced disease state in adult rats.</a> BMC Neuroscience. 17 (1), 69 (2016).</li><li>Xu, L., 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=CMV-beta-actin+promoter+directs+higher+expression+from+an+adeno-associated+viral+vector+in+the+liver+than+the+cytomegalovirus+or+elongation+factor+1+alpha+promoter+and+results+in+therapeutic+levels+of+human+factor+X+in+mice.">CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice.</a> Hum Gene Ther. 12 (5), 563-573 (2001).</li><li>Jackson, K., Dayton, R., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+vector+&amp;#34;magic+bullet&amp;#34;:+targeted+expression+in+the+central+nervous+system+after+peripheral+delivery+using+the+synapsin+promoter.">Gene vector &amp;#34;magic bullet&amp;#34;: targeted expression in the central nervous system after peripheral delivery using the synapsin promoter.</a> Expert Opin Ther Targets. 20 (10), 1153-1154 (2016).</li><li>van Hulst, R., Klein, J., Lachmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+embolism:+pathophysiology+and+treatment.">Gas embolism: pathophysiology and treatment.</a> Clin. Physiol. Funct. Imaging. 23 (5), 237-246 (2003).</li></ol>

En bred vifte af levering ruter er blevet testet for AAV vektorer: intra-parenkymalt, intra-cerebroventricular, intra-thecal, intravenøs, intranasal, intramuskulaer, osv. Hver rute kan have specifikke fordele samt ulemper. Hale vene administration af AAV til voksne rotter er en pålidelig metode til at opnå ensartede transduktion af CNS. Den perifere injektion metode undgår indførelsen af en injektion kanyle i CNS væv og er derfor minimalt invasiv. Effektiv CNS-udtryk kan opnås ved denne metode er den største ford...

Rekombinant adeno-associeret virus (AAV) vektorer er uundværlige redskaber til CNS forskning, fordi de er så effektive for transducing neuroner in vivo. AAV vektorerne er meget alsidige for at studere forskellige transgener og protein isoformer, forskellige væv, forskellige vært arter og forskellige indgiftsmåder. For eksempel, kan AAV administreres til mus af eksternt udstyr, relativt non-invasiv, intravenøs injektion til transduce neuroner i CNS som først beskrevet i Foust et al. og Duque et al. (Se JOVE papir af Gombash et al. (2014)). 1 , 2 , 3 dette gen levering tilgang anvendes i rotter til at effektivt udtrykke enten grøn fluorescerende proteiner (NGL) eller ALS relateret protein, transactive svar DNA-bindende protein af 43 kDa (TDP-43) i CNS. 4 , 5 , 6 , 7 , 8 , 9 arbejder i rotter er betydelig fordi rottens fysiologisk og metaboliske parametre er tættere på mennesker i forhold til mus og adfærdsmæssige og toksikologiske assays designet specielt til rotter. Desuden flere transgene rotte linjer bliver tilgængelige, kan udnyttes i AAV gen overførsel undersøgelser.
Metoder er beskrevet for ekspansiv CNS genoverførsel i rotter, og hurtig, pålidelig kvantificering af resultaterne. Omfattende CNS transduktion bruges til at efterligne symptomatologi af ALS i rotter ved at udtrykke TDP-43 i rygmarven. Metoden er hale vene injektioner af TDP-43 vektor til unge voksne rotter som anvendt i Jackson et al. 6 , 8 , 9 efter flere uger, TDP-43-induceret motor underskud er scoret af to metoder: undslippe rotarod som bruges i Dayton et al. og Jackson et al. og refleks 5 , 6 , 7 , 9 for kontrol normal god landbrugspraksis vektor, post mortem-analyse, den fluorescerende område af lillehjernen er beregnet som indekstal af transduktion effektivitet som bruges i Jackson et al. 6 , 8 analysen af lillehjernen har vist sig for at være en hurtig og pålidelig indeks af graden af CNS transduktion efter perifer gen levering og bør gælde for en række forskellige tilgange forsøger perifere til central genoverførsel.

<table>
	<tbody>
		<tr>
			<td>Scale</td>
			<td>Pelouze</td>
			<td>SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene bucket</td>
			<td>Thermo Scientific</td>
			<td>12014000</td>
			<td>4000 mL</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactated Ringer&#39;s Solution</td>
			<td>Baxter Healthcare</td>
			<td>0338-0114-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini vortexer</td>
			<td>Fisher Scientific</td>
			<td>128101</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>22624415</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory film</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>1-mL syringe</td>
			<td>BD</td>
			<td>309626</td>
			<td>Comes with a 25g needle attached. 
			Replace with 27-30g needle for injection</td>
		</tr>
		<tr>
			<td>30g needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>66794-013-25</td>
			<td>Isoflurane USP 250 mL</td>
		</tr>
		<tr>
			<td>Anesthesia mask</td>
			<td>Kopf Instruments</td>
			<td>906</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Henry Schein</td>
			<td>100-2524</td>
			<td>2&#34; x 2&#34; (5cm x 5cm)</td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>USA Scientific</td>
			<td>1111-0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipet</td>
			<td>Gilson</td>
			<td>4642080</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas anesthesia vaporizer</td>
			<td>SurgiVet</td>
			<td>4214322</td>
			<td>Classic T3</td>
		</tr>
		<tr>
			<td>Gas anesthesia scavenger</td>
			<td>SurgiVet</td>
			<td>32373B10</td>
			<td>Enviro-PURE</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Sears</td>
			<td>17280</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench pad</td>
			<td>VWR</td>
			<td>56617-006</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotarod</td>
			<td>Panlab/Harvard Apparatus</td>
			<td>76-0772</td>
			<td>4 rats/ 4 mice</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Decon Laboratories, Inc.</td>
			<td>2401</td>
			<td>140 proof</td>
		</tr>
		<tr>
			<td>70% Isopropanol</td>
			<td>VWR</td>
			<td>89108-160</td>
			<td></td>
		</tr>
		<tr>
			<td>Scion Image</td>
			<td>Scion Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GFP Tag Polyclonal Antibody</td>
			<td>Invitrogen</td>
			<td>A11122</td>
			<td>Primary Antibody - 1:500 dilution</td>
		</tr>
		<tr>
			<td>AlexaFluor 488</td>
			<td>Invitrogen</td>
			<td>A11070</td>
			<td>Secondary Antibody - 1:300 dilution</td>
		</tr>
		<tr>
			<td>Axioskop 40 microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

methods and tips for intravenous administration of adeno-associated virus to rats and evaluation of central nervous system transduction

Adeno-associeret virus (AAV) vektorer er en nøglen reagens i neurovidenskab for grupperet regelmæssigt interspaced korte palindromiske gentagelser (CRISPR), optogenetics, cre-lox målretning, osv. Formålet med dette manuskript er at støtte investigator forsøger ekspansiv centralnervesystemet (CNS) genoverførsel i rotten via hale vene injektion af AAV. Omfattende udtryk er relevante for betingelserne med udbredt patologi, og en rotte model er væsentlig på grund af dets større størrelse og fysiologisk ligheder til mennesker i forhold til mus. I dette eksempel program, er en omfattende neuronal transduktion bruges til at efterligne en neurodegenerativ sygdom, der påvirker hele rygmarven, amyotrofisk lateral sklerose (ALS). Effektiv omfattende CNS transduktion kan også bruges til at levere terapeutiske protein faktorer i prækliniske studier. Efter en post injektion udtryk interval af flere uger, er at evaluere virkningerne af transduktion. For en grøn fluorescerende proteiner (NGL) kontrol vektor skønnes mængden af normal god landbrugspraksis i lillehjernen hurtigt og pålideligt med et grundlæggende billedbehandlingsprogram. For motor sygdom fænotyper, der er fremkaldt af ALS relaterede protein transactive svar DNA-bindende protein af 43 kDa (TDP-43), scorede underskuddene af undslippe reflex og rotarod. Ud over sygdom modellering og genterapi er der forskellige potentielle anvendelsesmuligheder for omfattende-gen målretning beskrevet her. Udvidet brug af denne metode vil støtte i at fremskynde hypotesetest i neurovidenskab og neurogenetics.

TDP-43 induceret motor funktionshæmninger skulle opstå inden for 2-6 uger afhængig af dosis af den AAV TDP-43 anvendes (figur 1). Mislykket hale vene injektioner vil resultere i delvis eller ingen handicap; AAV skal nå blodbanen til at producere effekten. En vellykket injektion af AAV GFP vil resultere i normal god landbrugspraksis udtryk i hele hjernen og rygmarven i en vektor-dosis afhængige måde. Når du bruger stærke rekombinant initiativtageren ti...

Watch this Scientific Journal Video about Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction at JoVE.com

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Metoder og Tips til intravenøs indgift af Adeno-associeret Virus at rotter og evaluering af centralnervesystemet transduktion

Metoder til en omfattende centralnervesystemet gen levering i rotten er dækket. I dette eksempel er formålet at efterligne en sygdom, der påvirker hele rygmarven. Den udbredte transduktion kan bruges til at levere en terapeutisk protein til CNS fra en engangs, perifere administration.

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

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Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

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In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

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The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...