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

Forfatterne takker støtten fra Centro Multidisciplinar para Investigação Biológica na Área da Ciência em Animais de Laboratório (Cemib), Unicamp, til generering af AdipoSPH-mus, Inmmunometabolism and Cell Signaling Laboratory og National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) for al eksperimentel støtte. Vi takker den økonomiske støtte fra Sao Paulo Research Foundation (FAPESP): 2019/15025-5; 2020/09308-1; 2020/14725-0; 2021/11841-2.

Dyreforsøg blev udført i overensstemmelse med University of Campinas Guide for the Care and Use of Laboratory Animals (protokol CEUA #5810-1/2021).
1. Molekylær kloning
<ol><li>Design af single guide RNA'er (sgRNA'er)<ol><li>Design sgRNA'er til CRISPR-aktivering ved hjælp af CHOPCHOP, tilgængelig på https://chopchop.cbu.uib.no/, eller ethvert andet passende værktøj. Brug følgende parametre til at designe sgRNA rettet mod Prdm16-genet: Mål: Prdm16; I: Mus musculus; ...

<ol>
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En af de mest nyttige ikke-redigeringsanvendelser af CRISPR-teknologi er forhør af genfunktion gennem aktivering af endogene gener ved hjælp af CRISPRa-systemer6. SPH er en kraftfuld CRISPRa, der oprindeligt blev beskrevet for at inducere omdannelsen af astrocytter til aktive neuroner ved at målrette mod flere neurogene gener14. I denne undersøgelse blev AdipoSPH påvist at være et egnet værktøj til at undersøge beige fedtbiologi ved at aktivere ekspressionen af end...

Beige (eller brite) adipocytter afkobler protein 1 (UCP1)-ekspressive og mitokondrierige adipocytter, der befinder sig i hvide fedtvæv (WAT) depoter. Beige fedt fremkommer fra en delmængde af adipocytforfædre eller modne hvide adipocytter som reaktion på kuldeeksponering og andre stimuli 1,2. Beige adipocytter kan omdanne energi til varme på en UCP1-afhængig eller uafhængig måde3. Uanset dets termogene funktion kan beige fedt også forbedre metabolisk sundhed på andre måder, såsom udskillelse af adipokiner og antiinflammatoriske og antifibrotiske aktiviteter. Undersøgelser hos mus og mennesker har vist, at induktion af beige fedt forbedrer helkropsglukose og lipidhomeostase3. Men selvom vores viden om beige fedtbiologi har udviklet sig hurtigt i de senere år, er de fleste af dens metaboliske fordele og relaterede mekanismer stadig ikke fuldt ud forstået.
Grupperede regelmæssigt interspaced korte palindromiske gentagelser (CRISPR) blev først beskrevet i eukaryote celler som et værktøj, der er i stand til at generere et dobbeltstrengsbrud (DSB) på et specifikt sted i genomet gennem nukleaseaktiviteten af Cas9-proteinet 4,5. Cas9 styres af et syntetisk single-guide RNA (sgRNA) til at målrette mod en specifik genomisk region, hvilket fører til en DNA DSB. Ud over at bruge nukleasen Cas9 til redigeringsformål har CRISPR-Cas9-teknologien udviklet sig til at blive brugt som et sekvensspecifikt genreguleringsværktøj6. Udviklingen af et katalytisk inaktivt Cas9-protein (dCas9) og foreningen af transkriptionsmoduler, der er i stand til at forbedre genekspression, har givet anledning til CRISPR-aktiveringsværktøjer (CRISPRa). Flere CRISPRa-systemer er dukket op, såsom VP647,8, synergistisk aktiveringsmediator (SAM)9, SunTag10,11, VPR12,13 og SunTag-p65-HSF1 (SPH)14, som kombinerer komponenterne i SAM- og SunTag-aktivatorer. Det er for nylig blevet påvist, at den inducerede ekspression af neurogene gener i N2a-neuroblaster og primære astrocytter er højere ved hjælp af SPH sammenlignet med andre CRISPRa-systemer14, hvilket viser SPH som et lovende CRISPRa-værktøj.
Her udnyttede vi en tidligere udviklet SPH-mus14 til at generere en betinget musemodel, der udtrykker SPH specifikt i adipocytter ved hjælp af adiponectin Cre-afstamningen (AdipoSPH). Ved hjælp af en adeno-associeret virus (AAV), der bærer gRNA'et rettet mod det endogene Prdm16-gen, blev brunfarvning (hvid til beige konvertering) af inguinal WAT (iWAT) induceret for at øge ekspressionen af det termogene genprogram. Desuden øgede in vitro Prdm16 overekspression iltforbruget. Derfor giver denne protokol en alsidig SPH-musemodel til at udforske mekanismerne for beige fedtudvikling i fedtvæv.

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			<td>3,3&#39;,5-Triiodo-L-thyronine</td>
			<td>Sigma-Aldrich</td>
			<td>T2877</td>
			<td></td>
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			<td>3-Isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td></td>
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			<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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			<td>Dulbecco&#39;s Modification of Eagles Medium (DMEM)</td>
			<td>Corning</td>
			<td>10-017-CV</td>
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			<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>
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			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td></td>
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			<td>Excelta Self-Opening Micro Scissors</td>
			<td>Fisher Scientific</td>
			<td>17-467-496</td>
			<td></td>
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			<td>Fetal bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
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			<td>Fisherbrand Cell Scrapers (100 pk)</td>
			<td>Fisher Scientific</td>
			<td>08-100-241</td>
			<td></td>
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			<td>Fisherbrand High Precision Straight Tapered Ultra Fine Point Tweezers/Forceps</td>
			<td>Fisher Scientific</td>
			<td>12-000-122</td>
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			<td>Fisherbrand Sharp-Pointed Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-940</td>
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			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</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>
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			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378</td>
			<td></td>
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			<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>
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			<td>Maxiprep purification kit&#160;</td>
			<td>Qiagen</td>
			<td>12162</td>
			<td></td>
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			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td>Model 701</td>
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			<td>NEB 10-beta/Stable&#160;</td>
			<td>New England Biolabs</td>
			<td>C3019H</td>
			<td>E. coli competent cells</td>
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			<td>pAAV2/8&#160;</td>
			<td>Addgene&#160;</td>
			<td>112864</td>
			<td></td>
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			<td>pAAV-U6-gRNA-CBh-mCherry</td>
			<td>Addgene&#160;</td>
			<td>91947</td>
			<td></td>
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			<td>pAdDeltaF6&#160;</td>
			<td>Addgene&#160;</td>
			<td>112867</td>
			<td></td>
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			<td>PEG 8000</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
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			<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>
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			<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>
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			<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

Clustered regelmæssigt interspaced korte palindromiske gentagelser (CRISPR) teknologi har medført en revolution i biologi, og nyere værktøjer er blevet anvendt langt ud over den oprindeligt beskrevne genredigering. CRISPR-aktiveringssystemet (CRISPRa) kombinerer det katalytisk inaktive Cas9 (dCas9) protein med forskellige transkriptionsmoduler for at inducere endogen genekspression. SunTag-p65-HSF1 (SPH) er en nyudviklet CRISPRa-teknologi, der kombinerer komponenter fra synergistiske aktiveringsmediatorer (SAM'er) med SunTag-aktivatorerne. Dette system tillader overekspression af enkelte eller flere gener ved at designe et tilpasset single-guide RNA (sgRNA). I denne undersøgelse blev en tidligere udviklet SPH-mus brugt til at generere en betinget mus, der udtrykker SPH i adipocytter (adiponectin Cre-afstamning), kaldet AdipoSPH. For at inducere en hvid-til-beige fedt (bruning) fænotype blev en adeno-associeret virus (AAV), der bærer sgRNA rettet mod det endogene Prdm16-gen (en veletableret transkriptionsfaktor relateret til brun og beige fedtudvikling) injiceret i det inguinale hvide fedtvæv (iWAT). Denne musemodel inducerede ekspression af endogene Prdm16 og aktiverede det termogene genprogram. Desuden forbedrede in vitro SPH-induceret Prdm16-overekspression iltforbruget af beige adipocytter, fænokopiering af resultaterne af en tidligere Prdm16 transgen musemodel. Således beskriver denne protokol en alsidig, omkostningseffektiv og tidseffektiv musemodel til undersøgelse af fedtvævsbiologi.

AdipoSPH-mus blev udviklet ved avl af SPH- og Adipoq-Cre-musestammer. Begge musestammer var i en hybrid C57BL6J-DBA/2J baggrund (ifølge den kommercielle leverandør; se materialetabel). SPH-musens afstamning blev oprindeligt beskrevet af Zhou et al.14.
In vivo beige adipocytudvikling gennem AdipoSPH-medieret Prdm16-overekspression For at evaluere kapaciteten af modellen beskrevet i denne undersøgelse til a...

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

Denne protokol præsenterer brugen af CRISPR SunTag-p65-HSF1 (SPH) i adipocytter (AdipoSPH) som en alternativ strategi til adeno-associeret virus (AAV) til undersøgelse af beige fedtbiologi. In vivo-injektion af AAV-bærende sgRNA rettet mod det endogene Prdm16-gen er tilstrækkeligt til at inducere beige fedtudvikling og forbedre det termogene genprogram.

Undersøgelse af beige fedtbiologi og stofskifte ved hjælp af CRISPR SunTag-p65-HSF1 aktiveringssystemet

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

Denne protokol præsenterer SPH CRISPR-aktiveringssystemet som en alternativ strategi til konventionelle virale vektorer til at udføre forstærkningsfunktionsassays i adipocytter. Det tillader overekspression af enkelte eller flere adipocytendogene gener inden for fedtvævets komplekse cellulære miljø ved at levere et tilpasset enkeltguide-RNA ved hjælp af en adeno-associeret virus. De udfordrende trin i denne protokol er relateret til kloning af enkeltguide-RNA'et og produktion og injektion af adeno-associeret virus i fedtvævet.Til at begynde med skal du designe single guide RNA eller sgRNA til CRISPR-aktivering ved hjælp af chopchop eller ethvert andet passende værktøj. Design sgRNA'et målrettet PRDM16-genet ved hjælp af følgende parametre: Target som PRDM16 i en Mus musculus ved hjælp af en CRISPR / Cas9 og som aktivering. Tilføj udhæng til sgRNA'et for at matche Sac1-begrænsningsstedet i vektorrygraden PAAVU6GRNACBHM-kirsebær.Inkluder 5'AGCT 3'hvor n svarer til nukleotider. Få 3'sgRNA omvendt komplementsekvens ved hjælp af det angivne værktøj. Dernæst tilsættes en mikroliter af hver 5'og 3''enkeltstrenget oligonukleotid, en mikroliter T4-ligasebuffer, 0,5 mikroliter T4-polynukleotidkinase og 6,5 mikroliter vand til et slutreaktionsvolumen på 10 mikroliter.Anneal de gratis enkeltstrengede oligonukleotider ved hjælp af en termocyklist ved 37 grader Celsius i 30 minutter og 95 grader Celsius i fem minutter, efterfulgt af en rampehastighed på fem grader Celsius pr. Minut. Derefter tilsættes 25 nanogram plasmid PAAVU6GRNACBHM kirsebær til to mikroliter udglødede sgRNA-oligonukleotider til ligering af udglødede sgRNA-oligonukleotider, en mikroliter Sac1-enzym, to mikroliter 10X T4 DNA-ligasebuffer, en mikroliter T4-DNA-ligase og vand til et slutreaktionsvolumen på 10 mikroliter. Brug en termocyklist til at udføre ligering ved at inkubere reaktionsblandingen i 15 cyklusser ved 37 grader Celsius i fem minutter og 25 grader Celsius i fem minutter efterfulgt af at holde ved fire grader Celsius.Derefter transformeres de kompetente Escherichia coli DH10B-celler med fire mikroliter ligeringsprodukt ved varmechok ved 42 grader Celsius i 45 sekunder og spredes på en agarplade indeholdende 10 mikrogram pr. Milliliter Ampicillin. Bekræft transformerede kolonier ved Colony Polymerase Chain Reaction eller PCR ved hjælp af PCR Master Mix, vælg kolonien og bland den med fem mikroliter Master Mix, 0,1 mikroliter universel primer, 0,1 mikroliter sgRNA omvendt primer og fem mikroliter vand. Kør PCR ved hjælp af indledende denaturering ved 94 grader Celsius i to minutter, efterfulgt af 35 cyklusser med denaturering ved 94 grader Celsius i 20 sekunder, glødning i 30 sekunder ved 60 grader Celsius, forlængelse ved 72 grader Celsius i 30 sekunder og et sidste forlængelsestrin ved 72 grader Celsius i fem minutter.Efter PCR opløses DNA'et ved hjælp af agarosegelelektroforese i 0,5X TAE-buffer ved 90 volt i 30 minutter. Indsend positive prøver til Sanger-sekventering ved hjælp af universel primer. Rens derefter plasmidet fra en positiv klon ved hjælp af et plasmidrensningssæt efter producentens anvisninger.Placer den bedøvede mus i liggende stilling og barber et lille område på flankerne proksimalt til hofteleddet til inguinal hvidt fedtvæv eller IWAT-injektioner med en barbermaskine. Tilsæt hårfjerningscreme i fem minutter. Fjern resterende creme med vand for at undgå hudforbrændinger.Desinficere huden ved hjælp af tre skiftende runder med påføring af povidon jodopløsning på huden med rent gasbind og 70% alkohol. Lav derefter et snit på en til to centimeter med steriliseret saks i leddets proksimale område og hold huden åben ved hjælp af tang for at udsætte fedtdepotet. WAT kan fastgøres til huden på begge sider og strækker den fra begyndelsen fra ryggen og mod testiklerne.Brug pincet til forsigtigt at trække fedtdepotet opad gennem snittet for at sikre, at injektionen er på det rigtige sted og dybde. Fyld mikrolitersprøjten med 2,5 mikroliter adenoassocieret virus eller en AAV indeholdende sgRNA'et rettet mod det endogene PRDM16-gen. Indsæt forsigtigt nålen i en vinkel på 30-45 grader i IWAT.Gentag injektionen fem gange forskellige steder i vævet for homogent at inficere hele IWAT fedtpude. Placer musen på varmepuden, indtil bevidstheden er genvundet. Overvåg dyret hvert 10. til 15. minut, indtil det er helt genoprettet.Når dyret genvinder bevidstheden, skal du observere lokomotivprofilen, som skal være lineær og ikke have tegn på nød eller smerte. Se de stromale vaskulære celler eller SVF'er afledt af adipo SPH mus IWAT i en 6-brønds plade indeholdende komplet DMEM-medium i en til to timer, aspirer mediet, vask brønden to gange ved hjælp af PBS og erstat den med frisk, komplet medium. Inkuber cellerne ved 37 grader Celsius, 5% kuldioxid og 95% fugtighed, indtil cellerne når 70-80% sammenløb.På dag 0 induceres differentiering ved at behandle cellerne med induktionsmediet, og efter to dage erstattes induktionsmediet med vedligeholdelsesmediet. Igen efter to dage, på dag fire, skal du udskifte vedligeholdelsesmediet med et nyt vedligeholdelsesmedium i 2-3 dage. Skift vedligeholdelsesmediet hver 48. time, indtil preadipocytterne er fuldt differentieret til adipocytter.Overhold de modne adipocytter ved hjælp af lysmikroskopi, da de differentierede celler ser ud til at være fyldt med lipiddråber. Efter dyrkning af cellerne på en 6-brønds kulturplade med det komplette medium, indtil cellerne når 70-80% sammenløb, blandes 5,6 * 10 ^ 10 viralt genom pr. Mikroliter AAV-bærende sgRNA PRDM16 med to milliliter komplet medium og hexadimethrinbromid. Transducer cellerne ved at erstatte det komplette medium og tilføje det komplette medium, der indeholder AAV.Inkuber de transducerede celler i 12 timer ved 37 grader Celsius, 95% fugtighed og 5% kuldioxid. Opdel og se cellerne som beskrevet for celleproliferation og differentiering i beige adipocytter. Immunfluorescensbillederne fra IWAT af adipo SPH-mus viste ekspressionen af dCas9 i både kontrol- og PRDM16-gruppen.SPH-induceret PRDM16-ekspression inducerede klart en udbredt akkumulering af multiokulære beige adipocytter i IWAT. Desuden afslørede kvantitativ PCR en øget ekspression af PRDM16 og det termogene genprogram i PRDM16-gruppen sammenlignet med kontrolgruppen. Tilsvarende bekræftede in vitro-genekspressionsanalyse den øgede ekspression af det endogene PRDM16-gen og termogene gener i PRDM16-overekspressionsgruppen sammenlignet med kontrolgruppen.Den SPH-inducerede PRDM16-ekspression resulterede i højere basalt og maksimalt iltforbrug end i kontrolceller. Det er vigtigt, at dataene indikerede øget ukoblet respiration i PRDM16-gruppen sammenlignet med den i kontrolgruppen. Adipo SPH-modellen er velegnet til at undersøge flere gener og regulatoriske elementer inden for adipocytter.Denne metode åbner muligheden for en dybere forståelse af beige fedtbiologi.

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.

Bakteriel Detection og identifikation Brug elektrokemiske sensorer

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.

Anatomiske rekonstruktioner af Human Cardiac Venøs System ved hjælp Kontrast-computertomografi af Perfusion-faste Prøver

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.

Eksperimentel undersøgelse af Sekundære Flow Structures Downstream af en model type IV Stent Fejl i en 180 ° Buet Artery Test Sektion

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.

Stillads-understøttet transplantation af islænder i epididymisk fedtpude af diabetiske mus

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.

Brug Syntetisk Biologi Engineer levende celler der Grænseflade med 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 ...

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.

Effektiv Generation og redigering af Feeder-gratis IPSCs fra pancreas celler ved hjælp af 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 ...

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-hjerte side-fyring lys kateter til overvågning cellulære metabolisme ved hjælp af Transmural absorbans spektroskopi af Perfamale pattedyr hjerter

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 Genome redigering af Saccharomyces cerevisiae og skabelsen af gær pixel kunst

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.

Opførelse af CRISPR Plasmids og påvisning af Knockout Effektivitet i pattedyrceller gennem en Dobbelt 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.

Måling af 3D In-vivo Skulder kinematics ved hjælp af 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, ...