Name: investigation of beige fat biology and metabolism using the crispr suntag-p65-hsf1 activation system
Uploaded: 2023-01-06T14:20:00
Description: Watch this Scientific Journal Video about Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System at JoVE.com

De auteurs danken de steun van Centro Multidisciplinar para Investigação Biológica na Área da Ciência em Animais de Laboratório (Cemib), Unicamp, voor de generatie van AdipoSPH-muizen, het Inmmunometabolism and Cell Signaling Laboratory en het National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) voor alle experimentele ondersteuning. We danken de financiële steun van Sao Paulo Research Foundation (FAPESP): 2019/15025-5; 2020/09308-1; 2020/14725-0; 2021/11841-2.

Dierstudies werden uitgevoerd in overeenstemming met de University of Campinas Guide for the Care and Use of Laboratory Animals (protocol CEUA #5810-1/2021).
1. Moleculair klonen
<ol><li>Ontwerp van single guide RNA's (sgRNA's)<ol><li>Ontwerp sgRNA's voor CRISPR-activering met CHOPCHOP, verkrijgbaar bij https://chopchop.cbu.uib.no/, of een ander geschikt hulpmiddel. Gebruik de volgende parameters om sgRNA te ontwerpen dat gericht is op het Prdm16-gen: Target: Prdm16; In: Mus...

<ol>
	<li>Wang, W., Seale, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+brown+and+beige+fat+development.">Control of brown and beige fat development.</a> Nature Reviews Molecular Cell Biology. 17 (11), 691-702 (2016).</li><li>Wu, J., 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=Beige+adipocytes+are+a+distinct+type+of+thermogenic+fat+cell+in+mouse+and+human.">Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human.</a> Cell. 150 (2), 366-376 (2012).</li><li>Cohen, P., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+and+functional+complexity+of+thermogenic+fat.">The cellular and functional complexity of thermogenic fat.</a> Nature Reviews Molecular Cell Biology. 22 (6), 393-409 (2021).</li><li>Cong, 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=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Mali, P., 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=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li><li>Dominguez, A. A., Lim, W. A., Qi, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+editing:+Repurposing+CRISPR-Cas9+for+precision+genome+regulation+and+interrogation.">Beyond editing: Repurposing CRISPR-Cas9 for precision genome regulation and interrogation.</a> Nature Reviews Molecular Cell Biology. 17 (1), 5-15 (2016).</li><li>Gilbert, L. A., 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=CRISPR-mediated+modular+RNA-guided+regulation+of+transcription+in+eukaryotes.">CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.</a> Cell. 154 (2), 442-451 (2013).</li><li>Perez-Pinera, P., 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=RNA-guided+gene+activation+by+CRISPR-Cas9-based+transcription+factors.">RNA-guided gene activation by CRISPR-Cas9-based transcription factors.</a> Nature Methods. 10 (10), 973-976 (2013).</li><li>Konermann, 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=Genome-scale+transcriptional+activation+by+an+engineered+CRISPR-Cas9+complex.">Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.</a> Nature. 517 (7536), 583-588 (2015).</li><li>Gilbert, L. A., 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=Genome-scale+CRISPR-mediated+control+of+gene+repression+and+activation.">Genome-scale CRISPR-mediated control of gene repression and activation.</a> Cell. 159 (3), 647-661 (2014).</li><li>Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S., Vale, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein-tagging+system+for+signal+amplification+in+gene+expression+and+fluorescence+imaging.">A protein-tagging system for signal amplification in gene expression and fluorescence imaging.</a> Cell. 159 (3), 635-646 (2014).</li><li>Chavez, A., 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=Highly+efficient+Cas9-mediated+transcriptional+programming.">Highly efficient Cas9-mediated transcriptional programming.</a> Nature Methods. 12 (4), 326-328 (2015).</li><li>Chavez, A., 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=Comparison+of+Cas9+activators+in+multiple+species.">Comparison of Cas9 activators in multiple species.</a> Nature Methods. 13 (7), 563-567 (2016).</li><li>Zhou, H., 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=In+vivo+simultaneous+transcriptional+activation+of+multiple+genes+in+the+brain+using+CRISPR-dCas9-activator+transgenic+mice.">In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice.</a> Nature Neuroscience. 21 (3), 440-446 (2018).</li><li>Fripont, S., Marneffe, C., Marino, M., Rincon, M. Y., Holt, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+purification,+and+quality+control+for+adeno-associated+virus-based+vectors.">Production, purification, and quality control for adeno-associated virus-based vectors.</a> Journal of Visualized Experiments. (143), e58960 (2019).</li><li>Negrini, M., Wang, G., Heuer, A., Bj&#246;rklund, T., Davidsson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+production+everywhere:+a+simple,+fast,+and+reliable+protocol+for+in-house+aav+vector+production+based+on+chloroform+extraction.">AAV production everywhere: a simple, fast, and reliable protocol for in-house aav vector production based on chloroform extraction.</a> Current Protocols in Neuroscience. 93 (1), 103 (2020).</li><li>Aune, U. L., Ruiz, L., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+differentiation+of+stromal+vascular+cells+to+beige/brite+cells.">Isolation and differentiation of stromal vascular cells to beige/brite cells.</a> Journal of Visualized Experiments. (73), e50191 (2013).</li><li>Wang, Q., 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=Post-translational+control+of+beige+fat+biogenesis+by+PRDM16+stabilization.">Post-translational control of beige fat biogenesis by PRDM16 stabilization.</a> Nature. 609 (7925), 151-158 (2022).</li><li>Oeckl, J., Bast-Habersbrunner, A., Fromme, T., Klingenspor, M., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+functional+analysis+of+murine+thermogenic+adipocytes.">Isolation, culture, and functional analysis of murine thermogenic adipocytes.</a> STAR Protocols. 1 (3), 100118 (2020).</li><li>Cohen, P., 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=Ablation+of+PRDM16+and+beige+adipose+causes+metabolic+dysfunction+and+a+subcutaneous+to+visceral+fat+switch.">Ablation of PRDM16 and beige adipose causes metabolic dysfunction and a subcutaneous to visceral fat switch.</a> Cell. 156 (1-2), 304-316 (2014).</li><li>Harms, M., Seale, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+and+beige+fat:+development,+function+and+therapeutic+potential.">Brown and beige fat: development, function and therapeutic potential.</a> Nature Medicine. 19 (10), 1252-1263 (2013).</li><li>Divakaruni, A. S., Jastroch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+for+the+analysis,+standardization+and+interpretation+of+oxygen+consumption+measurements.">A practical guide for the analysis, standardization and interpretation of oxygen consumption measurements.</a> Nature Metabolism. 4 (8), 978-994 (2022).</li><li>Seale, P., 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=Prdm16+determines+the+thermogenic+program+of+subcutaneous+white+adipose+tissue+in+mice.">Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice.</a> The Journal of Clinical Investigation. 121 (1), 96-105 (2011).</li><li>Valet, P., Tavernier, G., Castan-Laurell, I., Saulnier-Blache, J. S., Langin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+adipose+tissue+development+from+transgenic+animal+models.">Understanding adipose tissue development from transgenic animal models.</a> Journal of Lipid Research. 43 (6), 835-860 (2002).</li><li>Bates, R., Huang, W., Cao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue:+an+emerging+target+for+adeno-associated+viral+vectors.">Adipose tissue: an emerging target for adeno-associated viral vectors.</a> Molecular Therapy. Methods &amp; Clinical Development. 19, 236-249 (2020).</li><li>Wang, D., Tai, P. W. L., Guangping, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+vector+as+a+platform+for+gene+therapy+delivery.">Adeno-associated virus vector as a platform for gene therapy delivery.</a> Nature Reviews Drug Discovery. 18 (5), 358-378 (2019).</li><li>Colella, P., Ronzitti, G., Mingozzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+issues+in+AAV-mediated+in+vivo+gene+therapy.">Emerging issues in AAV-mediated in vivo gene therapy.</a> Molecular Therapy. Methods &amp; Clinical Development. 8, 87-104 (2018).</li><li>Deshmukh, A. 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=Proteomics-based+comparative+mapping+of+the+secretomes+of+human+brown+and+white+adipocytes+reveals+EPDR1+as+a+novel+batokine.">Proteomics-based comparative mapping of the secretomes of human brown and white adipocytes reveals EPDR1 as a novel batokine.</a> Cell Metabolism. 30 (5), 963-975 (2019).</li><li>Sponton, C. H., 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=The+regulation+of+glucose+and+lipid+homeostasis+via+PLTP+as+a+mediator+of+BAT-liver+communication.">The regulation of glucose and lipid homeostasis via PLTP as a mediator of BAT-liver communication.</a> EMBO reports. 21 (9), 49828 (2020).</li></ol>

Een van de meest bruikbare niet-bewerkende toepassingen van CRISPR-technologie is de ondervraging van de genfunctie door de activering van endogene genen met behulp van CRISPRa-systemen6. SPH is een krachtige CRISPRa die oorspronkelijk werd beschreven om de omzetting van astrocyten in actieve neuronen te induceren door zich te richten op verschillende neurogene genen14. In deze studie werd aangetoond dat AdipoSPH een geschikt hulpmiddel is voor het onderzoeken van de biolog...

Beige (of brite) adipocyten zijn ontkoppelende eiwit 1 (UCP1) -tot expressie brengende en mitochondriaal-rijke adipocyten die zich bevinden in depots van wit vetweefsel (WAT). Beige vet komt voort uit een subset van adipocytenvoorlopers of rijpe witte adipocyten als reactie op blootstelling aan koude en andere stimuli 1,2. Beige adipocyten kunnen energie omzetten in warmte op een UCP1-afhankelijke of onafhankelijke manier3. Ongeacht de thermogene functie kan beige vet ook de metabole gezondheid verbeteren door andere middelen, zoals de afscheiding van adipokines en ontstekingsremmende en anti-fibrotische activiteiten. Studies bij muizen en mensen hebben aangetoond dat de inductie van beige vet de glucose van het hele lichaam en de lipidehomeostase verbetert3. Hoewel onze kennis van beige vetbiologie de afgelopen jaren snel is geëvolueerd, zijn de meeste metabole voordelen en gerelateerde mechanismen nog steeds niet volledig begrepen.
Geclusterde regelmatig interspaced korte palindromische herhalingen (CRISPR) werden voor het eerst beschreven in eukaryote cellen als een hulpmiddel dat in staat is om een dubbelstrengsbreuk (DSB) te genereren op een specifieke plaats in het genoom door de nucleaseactiviteit van het Cas9-eiwit 4,5. Cas9 wordt geleid door een synthetisch single-guide RNA (sgRNA) om zich te richten op een specifiek genomisch gebied, wat leidt tot een DNA-DSB. Naast het gebruik van het nuclease Cas9 voor bewerkingsdoeleinden, is de CRISPR-Cas9-technologie geëvolueerd om te worden gebruikt als een sequentiespecifiek genregulatie-instrument6. De ontwikkeling van een katalytisch inactief Cas9-eiwit (dCas9) en de associatie van transcriptionele modules die de genexpressie kunnen verbeteren, heeft geleid tot CRISPR-activeringstools (CRISPRa). Er zijn verschillende CRISPRa-systemen ontstaan, zoals VP647,8, synergetische activeringsmediator (SAM)9, SunTag10,11, VPR12,13 en SunTag-p65-HSF1 (SPH)14, die de componenten van SAM- en SunTag-activatoren combineert. Onlangs is aangetoond dat de geïnduceerde expressie van neurogene genen in N2a-neuroblasten en primaire astrocyten hoger is met behulp van SPH in vergelijking met andere CRISPRa-systemen14, wat SPH aantoont als een veelbelovend CRISPRa-hulpmiddel.
Hier maakten we gebruik van een eerder ontwikkelde SPH-muis14 om een voorwaardelijk muismodel te genereren dat SPH specifiek in adipocyten tot expressie brengt met behulp van de adiponectine Cre-afstamming (AdipoSPH). Met behulp van een adeno-geassocieerd virus (AAV) dat het gRNA draagt dat zich richt op het endogene Prdm16-gen, werd bruining (witte naar beige conversie) van inguinale WAT (iWAT) geïnduceerd om de expressie van het thermogene genprogramma te verhogen. Bovendien verhoogde in vitro Prdm16-overexpressie het zuurstofverbruik. Daarom biedt dit protocol een veelzijdig SPH-muismodel voor het verkennen van de mechanismen van beige vetontwikkeling in vetweefsel.

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			<td>3,3&#39;,5-Triiodo-L-thyronine</td>
			<td>Sigma-Aldrich</td>
			<td>T2877</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td></td>
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		<tr>
			<td>AAVpro 293T Cell Line</td>
			<td>Takarabio</td>
			<td>632273</td>
			<td></td>
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			<td>Amicon Ultra Centrifugal Filter</td>
			<td>Merckmillipore</td>
			<td>UFC510008</td>
			<td>100 KDa</td>
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			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D1756</td>
			<td></td>
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		<tr>
			<td>Dulbecco&#39;s Modification of Eagles Medium (DMEM)</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) F-12, GlutaMAX&#8482; supplement</td>
			<td>Gibco</td>
			<td>10565-018</td>
			<td>high concentrations of glucose, amino acids, and vitamins</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>Excelta Self-Opening Micro Scissors</td>
			<td>Fisher Scientific</td>
			<td>17-467-496</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Cell Scrapers (100 pk)</td>
			<td>Fisher Scientific</td>
			<td>08-100-241</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand High Precision Straight Tapered Ultra Fine Point Tweezers/Forceps</td>
			<td>Fisher Scientific</td>
			<td>12-000-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Sharp-Pointed Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-940</td>
			<td></td>
		</tr>
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			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
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			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-25G</td>
			<td></td>
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			<td>Hexadimethrine bromide (Polybrene)</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td></td>
		</tr>
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			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378</td>
			<td></td>
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		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I9278</td>
			<td></td>
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			<td>LigaFast Rapid DNA Ligation System</td>
			<td>Promega</td>
			<td>M8225</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxiprep purification kit&#160;</td>
			<td>Qiagen</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td>Model 701</td>
		</tr>
		<tr>
			<td>NEB 10-beta/Stable&#160;</td>
			<td>New England Biolabs</td>
			<td>C3019H</td>
			<td>E. coli competent cells</td>
		</tr>
		<tr>
			<td>pAAV2/8&#160;</td>
			<td>Addgene&#160;</td>
			<td>112864</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-U6-gRNA-CBh-mCherry</td>
			<td>Addgene&#160;</td>
			<td>91947</td>
			<td></td>
		</tr>
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			<td>pAdDeltaF6&#160;</td>
			<td>Addgene&#160;</td>
			<td>112867</td>
			<td></td>
		</tr>
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			<td>PEG 8000</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Sigma-Aldrich</td>
			<td>23966</td>
			<td>Linear, MW 25000</td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Rioqu&#237;mica</td>
			<td>510101303</td>
			<td>Antiseptic</td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Sigma-Aldrich</td>
			<td>R2408</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI enzyme</td>
			<td>New England Biolabs</td>
			<td>R0156</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Design Premier Adson Forceps</td>
			<td>Fisher Scientific</td>
			<td>22-079-741</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>475-40417</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Promega</td>
			<td>M180B</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase buffer&#160;</td>
			<td>New England Biolabs</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK enzyme kit</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tramadol Hydrochloride</td>
			<td>SEM</td>
			<td>43930</td>
			<td></td>
		</tr>
		<tr>
			<td>Vidisic Gel&#160;</td>
			<td>Bausch + Lomb&#160;</td>
			<td>99620</td>
			<td></td>
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</table>

investigation of beige fat biology and metabolism using the crispr suntag-p65-hsf1 activation system

Geclusterde regelmatig interspaced short palindromic repeats (CRISPR) -technologie heeft een revolutie in de biologie teweeggebracht en recente hulpmiddelen zijn veel verder toegepast dan de oorspronkelijk beschreven genbewerking. Het CRISPR-activeringssysteem (CRISPRa) combineert het katalytisch inactieve Cas9 (dCas9)-eiwit met verschillende transcriptiemodules om endogene genexpressie te induceren. SunTag-p65-HSF1 (SPH) is een recent ontwikkelde CRISPRa-technologie die componenten van synergetische activeringsmediatoren (SAM's) combineert met de SunTag-activatoren. Dit systeem maakt de overexpressie van enkele of meerdere genen mogelijk door een aangepast single-guide RNA (sgRNA) te ontwerpen. In deze studie werd een eerder ontwikkelde SPH-muis gebruikt om een voorwaardelijke muis te genereren die SPH tot expressie brengt in adipocyten (adiponectine Cre-afstamming), genaamd AdipoSPH. Om een wit-naar-beige vet (bruining) fenotype te induceren, werd een adeno-geassocieerd virus (AAV) met sgRNA gericht op het endogene Prdm16-gen (een gevestigde transcriptiefactor gerelateerd aan de ontwikkeling van bruin en beige vet) geïnjecteerd in het inguinale witte vetweefsel (iWAT). Dit muismodel induceerde de expressie van endogene Prdm16 en activeerde het thermogene genprogramma. Bovendien verhoogde in vitro SPH-geïnduceerde Prdm16-overexpressie het zuurstofverbruik van beige adipocyten, waardoor de resultaten van een eerder Prdm16-transgeen muismodel werden gefenocopeerd. Dit protocol beschrijft dus een veelzijdig, kosteneffectief en tijdeffectief muismodel voor het onderzoeken van vetweefselbiologie.

AdipoSPH-muizen werden ontwikkeld door SPH- en Adipoq-Cre-muizenstammen te fokken. Beide muizenstammen hadden een hybride C57BL6J-DBA/2J-achtergrond (volgens de commerciële leverancier; zie Materiaaltabel). De SPH-muizenlijn werd oorspronkelijk beschreven door Zhou et al.14.
In vivo beige adipocytenontwikkeling door AdipoSPH-gemedieerde Prdm16-overexpressie Om de capaciteit van het in deze studie beschreven...

Watch this Scientific Journal Video about Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System at JoVE.com

Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System

Dit protocol presenteert het gebruik van CRISPR SunTag-p65-HSF1 (SPH) in adipocyten (AdipoSPH) als een alternatieve strategie voor adeno-geassocieerd virus (AAV) voor het onderzoeken van beige vetbiologie. In vivo injectie van AAV-dragend sgRNA gericht op het endogene Prdm16-gen is voldoende om de ontwikkeling van beige vet te induceren en het thermogene genprogramma te verbeteren.

Onderzoek naar beige vetbiologie en metabolisme met behulp van het CRISPR SunTag-p65-HSF1-activeringssysteem

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has prompted a revolution in biology, and recent tools have been applied far ...

Dit protocol presenteert het SPH CRISPR-activeringssysteem als een alternatieve strategie voor conventionele virale vectoren om gain-functietests uit te voeren in adipocyten. Het maakt de overexpressie van enkele of meerdere adipocyten endogene genen binnen de complexe cellulaire omgeving van vetweefsel mogelijk door een aangepast single guide RNA te leveren met behulp van een adeno-geassocieerd virus. De uitdagende stappen van dit protocol houden verband met het klonen van het single guide RNA en de productie en injectie van adeno-geassocieerd virus in het vetweefsel.Ontwerp om te beginnen single guide RNA of sgRNA voor CRISPR-activering met behulp van chopchop of een ander geschikt hulpmiddel. Ontwerp het sgRNA gericht op het PRDM16-gen met behulp van de volgende parameters:Target als PRDM16 in een Mus-musculus met behulp van een CRISPR / Cas9 en voor als activering. Voeg overhangen toe aan het sgRNA om overeen te komen met de Sac1-restrictieplaats in de vectorbackbone PAAVU6GRNACBHM-kers.Inclusief 5'AGCT 3', waarbij n overeenkomt met nucleotiden. Verkrijg 3'sgRNA reverse complement sequence met behulp van de aangegeven tool. Voeg vervolgens voor het gloeien van enkelstrengs complementaire oligonucleotiden één microliter van elk 5'en 3'enkelstrengs oligonucleotide, één microliter T4-ligasebuffer, 0,5 microliter T4-polynucleotidekinase en 6,5 microliter water toe voor een eindreactievolume van 10 microliter.Gloei de gratis enkelstrengs oligonucleotiden met behulp van een thermocycler bij 37 graden Celsius gedurende 30 minuten en 95 graden Celsius gedurende vijf minuten, gevolgd door een afbouwsnelheid van vijf graden Celsius per minuut. Voeg vervolgens voor ligatie van gegloeide sgRNA-oligonucleotiden 25 nanogram plasmide PAAVU6GRNACBHM-kers toe aan twee microliter gegloeid sgRNA Oligonucleotiden, één microliter Sac1-enzym, twee microliter 10X T4 DNA-ligasebuffer, één microliter T4 DNA-ligase en water tot een eindreactievolume van 10 microliter. Voer met behulp van een thermocycler ligatie uit door het reactiemengsel gedurende 15 cycli bij 37 graden Celsius gedurende vijf minuten en 25 graden Celsius gedurende vijf minuten te incuberen, gevolgd door vier graden Celsius vast te houden.Transformeer vervolgens de bevoegde Escherichia coli DH10B-cellen met vier microliter van het ligatieproduct door hitteschok bij 42 graden Celsius gedurende 45 seconden en verspreid op een agarplaat met 10 microgram per milliliter Ampicilline. Bevestig getransformeerde kolonies door Colony Polymerase Chain Reaction of PCR met behulp van de PCR Master Mix, kies de kolonie en meng deze met vijf microliter Master Mix, 0,1 microliter universele primer, 0,1 microliter sgRNA reverse primer en vijf microliter water. Voer de PCR uit met behulp van initiële denaturatie bij 94 graden Celsius gedurende twee minuten, gevolgd door 35 cycli van denaturatie bij 94 graden Celsius gedurende 20 seconden, gloeien gedurende 30 seconden bij 60 graden Celsius, extensie bij 72 graden Celsius gedurende 30 seconden en een laatste rekstap bij 72 graden Celsius gedurende vijf minuten.Los na PCR het DNA op met behulp van agarosegel-elektroforese in 0,5X TAE-buffer bij 90 volt gedurende 30 minuten. Dien positieve monsters in voor Sanger Sequencing met behulp van universele primer. Zuiver vervolgens het plasmide van een positieve kloon met behulp van een plasmidezuiveringskit volgens de instructies van de fabrikant.Plaats de verdoofde muis in rugligging en scheer een klein gebied op de flanken proximaal van de heupgewrichten voor inguinaal wit vetweefsel of IWAT-injecties met een scheerapparaat. Voeg vijf minuten ontharingscrème toe. Verwijder resterende crème met water om brandwonden op de huid te voorkomen.Desinfecteer de huid met behulp van drie afwisselende rondes van het aanbrengen van de povidon jodiumoplossing op de huid met schoon gaas en 70% alcohol. Maak vervolgens een incisie van één tot twee centimeter met een gesteriliseerde schaar in het proximale gebied van de gewrichten en houd de huid open met een tang om het vetdepot bloot te leggen. De WAT kan aan beide zijden aan de huid worden bevestigd en verlengt deze vanaf het begin van de achterkant en naar de teelballen.Trek met een tang het vetdepot voorzichtig omhoog door de incisie om ervoor te zorgen dat de injectie op de juiste locatie en diepte is. Vul de microliterspuit met 2,5 microliter van het adeno-geassocieerde virus of een AAV die het sgRNA bevat dat zich richt op het endogene PRDM16-gen. Steek de naald voorzichtig in een hoek van 30-45 graden in de IWAT.Herhaal de injectie vijf keer op verschillende locaties van het weefsel om het hele IWAT-vetkussen homogeen te infecteren. Plaats de muis op het verwarmingskussen totdat het bewustzijn is herwonnen. Controleer het dier elke 10 tot 15 minuten totdat het volledig is hersteld.Nadat het dier weer bij bewustzijn is gekomen, observeert u de locomotiefprofilering, die lineair moet zijn en geen tekenen van angst of pijn moet hebben. Zie de stromale vasculaire cellen of SVF's afgeleid van adipo SPH muis IWAT in een 6-Well plaat met volledig DMEM-medium gedurende één tot twee uur, zuig het medium op, was de put twee keer met PBS en vervang het door vers, compleet medium. Incubeer de cellen bij 37 graden Celsius, 5% koolstofdioxide en 95% vochtigheid totdat de cellen 70-80% samenvloeiing bereiken.Induceer op dag 0 differentiatie door de cellen te behandelen met het inductiemedium en vervang na twee dagen het inductiemedium door het onderhoudsmedium. Vervang na twee dagen, op dag vier, het onderhoudsmedium gedurende 2-3 dagen door een vers onderhoudsmedium. Vervang het onderhoudsmedium elke 48 uur totdat de preadipocyten volledig zijn gedifferentieerd in adipocyten.Observeer de volwassen adipocyten met behulp van lichtmicroscopie terwijl de gedifferentieerde cellen lijken te zijn geladen met lipidedruppels. Na het kweken van de cellen op een 6-Well kweekplaat met het volledige medium totdat de cellen 70-80% confluentie bereiken, meng 5,6 * 10 ^ 10 viraal genoom per microliter AAV-dragend sgRNA PRDM16 met twee milliliter compleet medium en hexadimethrinebromide. Transduceer de cellen door het volledige medium te vervangen en het volledige medium met AAV toe te voegen.Incubeer de getransduceerde cellen gedurende 12 uur bij 37 graden Celsius 95% vochtigheid en 5% koolstofdioxide. Splits en zie de cellen zoals beschreven voor celproliferatie en differentiatie in beige adipocyten. De immunofluorescentiebeelden van IWAT van adipo SPH-muizen toonden de expressie van dCas9 in zowel de controle- als de PRDM16-groep.SPH-geïnduceerde PRDM16-expressie veroorzaakte duidelijk een wijdverspreide accumulatie van multi-oculaire beige adipocyten in IWAT. Bovendien onthulde kwantitatieve PCR een verhoogde expressie van PRDM16 en het thermogene genprogramma in de PRDM16-groep in vergelijking met de controlegroep. Evenzo bevestigde in vitro genexpressieanalyse de verhoogde expressie van het endogene PRDM16-gen en thermogene genen in de PRDM16-overexpressiegroep in vergelijking met de controlegroep.De SPH-geïnduceerde PRDM16-expressie resulteerde in een hoger basaal en maximaal zuurstofverbruik dan dat in controlecellen. Belangrijk is dat de gegevens wezen op een verbeterde ontkoppelde ademhaling in de PRDM16-groep in vergelijking met die in de controlegroep. Het Adipo SPH-model is geschikt voor het onderzoeken van meerdere genen en regulerende elementen binnen adipocyten.Deze methode opent de mogelijkheid van een dieper begrip van de biologie van beige vet.

investigation-beige-fat-biology-metabolism-using-crispr-suntag-p65

Research

JoVE Journal

Bioengineering

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bacteriële Detection &amp; Identification gebruiken elektrochemische sensoren

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

Anatomische Reconstructies van de Mens Cardiac veneuze systeem met contrastdetectie computertomografie van perfusie-vaste Monsters

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and tortuosity). Secondary flow structures are vortical flow patterns that occur in curved arteries due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. Such flow morphologies are greatly affected by pulsatility and multiple harmonics of physiological inflow conditions and vary greatly in size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows 1 - 7.
Secondary flow structures may ultimately influence the wall shear stress and exposure time of blood-borne particles toward progression of atherosclerosis, restenosis, sensitization of platelets and thrombosis 4 - 6, 8 - 13. Therefore, the ability to detect and characterize these structures under laboratory-controlled conditions is precursor to further clinical investigations.
A common surgical treatment to atherosclerosis is stent implantation, to open up stenosed arteries for unobstructed blood flow. But the concomitant flow perturbations due to stent installations result in multi-scale secondary flow morphologies 4 - 6. Progressively higher order complexities such as asymmetry and loss in coherence can be induced by ensuing stent failures vis-&#224;-vis those under unperturbed flows 5. These stent failures have been classified as &#34;Types I-to-IV&#34; based on failure considerations and clinical severity 14.
This study presents a protocol for the experimental investigation of the complex secondary flow structures due to complete transverse stent fracture and linear displacement of fractured parts (&#34;Type IV&#34;) in a curved artery model. The experimental method involves the implementation of particle image velocimetry (2C-2D PIV) techniques with an archetypal carotid artery inflow waveform, a refractive index matched blood-analog working fluid for phase-averaged measurements 15 - 18. Quantitative identification of secondary flow structures was achieved using concepts of flow physics, critical point theory and a novel wavelet transform algorithm applied to experimental PIV data 5, 6, 19 - 26.

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

Experimenteel onderzoek van secundaire Flow Structures Stroomafwaarts van een Model Type IV Stent Niet in een 180 ° Gebogen Artery proefvak

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Steiger-gesteunde transplantatie van eilanden in het epididymale vetpaneel van diabetische muizen

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

Met behulp van synthetische biologie levende cellen Engineer die interface met programmeerbare Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Efficiënte generatie en het bewerken van Feeder-vrije IPSCs van menselijke Pancreatic cellen met behulp van de CRISPR-Cas9-systeem

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Intra-cardiale side-afvuren licht katheter voor het monitoren van cellulaire metabolisme met behulp van Transmural extinctie spectroscopie van Geperfectiongebruikt zoogdieren harten

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

CRISPR/Cas12a multiplex genoom bewerking van Saccharomyces cerevisiae en de creatie van gist pixel art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Here, we present a protocol describing a streamlined method for the efficient generation of plasmids expressing both the CRISPR enzyme and associated single guide RNA (sgRNAs). Co-transfection of mammalian cells with this sgRNA/CRISPR vector and a dual luciferase reporter vector that examines double-strand break repair allows evaluation of knockout efficiency.

Bouw van CRISPR Plasmids en detectie van knock-out efficiëntie in zoogdiercellen door middel van een Dual Luciferase Reporter System

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

Meten van 3D in-vivo schouderkinematica met behulp van biplanaire videoradiografie

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...