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

Yazarlar, Centro Multidisciplinar para Investigação Biológica na Área da Ciência em Animais de Laboratório (Cemib), Unicamp'tan AdipoSPH farelerinin üretimi için aldıkları destek, Inmmunometabolizma ve Hücre Sinyalizasyon Laboratuvarı ve Hücre Biyolojisine Uygulanan Fotonik Ulusal Bilim ve Teknoloji Enstitüsü'ne (INFABIC) tüm deneysel destek için teşekkür eder. Sao Paulo Araştırma Vakfı'nın (FAPESP) finansal desteğine teşekkür ederiz: 2019/15025-5; 2020/09308-1; 2020/14725-0; 2021/11841-2.

Hayvan çalışmaları, Campinas Üniversitesi Laboratuvar Hayvanlarının Bakımı ve Kullanımı Kılavuzu'na (protokol CEUA #5810-1/2021) uygun olarak gerçekleştirilmiştir.
1. Moleküler klonlama
<ol><li>Tek kılavuzlu RNA'ların (sgRNA'lar) tasarımı<ol><li>https://chopchop.cbu.uib.no/'de mevcut olan CHOPCHOP'u veya başka bir uygun aracı kullanarak CRISPR aktivasyonu için sgRNA'lar tasarlayın. Prdm16 genini hedefleyen sgRNA'yı tasarlamak için aşağıdaki param...

<ol>
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CRISPR teknolojisinin en kullanışlı düzenleme dışı uygulamalarından biri, CRISPRa sistemleri6 kullanılarak endojen genlerin aktivasyonu yoluyla gen fonksiyonunun sorgulanmasıdır. SPH, başlangıçta birkaç nörojenik geni hedef alarak astrositlerin aktif nöronlara dönüşümünü indüklediği açıklanan güçlü bir CRISPRa'dır14. Bu çalışmada, AdipoSPH'nin adipositlerde endojen Prdm16 ekspresyonunu aktive ederek bej yağ biyolojisini araştırmak için ...

Bej (veya brite) adipositler, beyaz yağ dokusu (WAT) depolarında bulunan protein 1 (UCP1) eksprese edici ve mitokondriyal bakımından zengin adipositleri ayırır. Bej yağ, soğuğa maruz kalma ve diğer uyaranlara yanıt olarak adiposit progenitörlerinin veya olgun beyaz adipositlerin bir alt kümesinden ortaya çıkar 1,2. Bej adipositler, enerjiyi UCP1'e bağımlı veya bağımsız bir şekilde ısıya dönüştürebilir3. Termojenik işlevinden bağımsız olarak, bej yağ, adipokinlerin salgılanması ve anti-enflamatuar ve anti-fibrotik aktiviteler gibi diğer yollarla metabolik sağlığı da iyileştirebilir. Farelerde ve insanlarda yapılan çalışmalar, bej yağın indüksiyonunun tüm vücut glikozunu ve lipit homeostazını iyileştirdiğini göstermiştir3. Bununla birlikte, bej yağ biyolojisi hakkındaki bilgimiz son yıllarda hızla gelişmesine rağmen, metabolik faydalarının çoğu ve ilgili mekanizmaları hala tam olarak anlaşılamamıştır.
Kümelenmiş düzenli aralıklı kısa palindromik tekrarlar (CRISPR) ilk olarak ökaryotik hücrelerde, Cas9 proteini 4,5'in nükleaz aktivitesi yoluyla genomdaki belirli bir bölgede çift iplikçik kırılması (DSB) üretebilen bir araç olarak tanımlanmıştır. Cas9, belirli bir genomik bölgeyi hedeflemek için sentetik bir tek kılavuzlu RNA (sgRNA) tarafından yönlendirilir ve bir DNA DSB'ye yol açar. Nükleaz Cas9'u düzenleme amacıyla kullanmanın yanı sıra, CRISPR-Cas9 teknolojisi, diziye özgü bir gen düzenleme aracı6 olarak kullanılmak üzere gelişmiştir. Katalitik olarak inaktif bir Cas9 proteininin (dCas9) geliştirilmesi ve gen ekspresyonunu artırabilen transkripsiyonel modüllerin ilişkisi, CRISPR aktivasyonu (CRISPRa) araçlarına yol açmıştır. SAM ve SunTag aktivatörlerinin bileşenlerini birleştiren VP647,8, sinerjik aktivasyon aracısı (SAM)9, SunTag10,11, VPR12,13 ve SunTag-p65-HSF1 (SPH)14 gibi çeşitli CRISPRa sistemleri ortaya çıkmıştır. Son zamanlarda, N2a nöroblastlarında ve primer astrositlerde nörojenik genlerin indüklenmiş ekspresyonunun, diğer CRISPRa sistemlerine kıyasla SPH kullanılarak daha yüksek olduğu gösterilmiştir14, SPH'yi umut verici bir CRISPRa aracı olarak göstermektedir.
Burada, adiponektin Cre soyunu (AdipoSPH) kullanarak SPH'yi özellikle adipositlerde ifade eden koşullu bir fare modeli oluşturmak için daha önce geliştirilmiş bir SPH fare14'ten yararlandık. Endojen Prdm16 genini hedef alan gRNA'yı taşıyan adeno ilişkili bir virüs (AAV) kullanılarak, termojenik gen programının ekspresyonunu arttırmak için inguinal WAT'ın (iWAT) kahverengileşmesi (beyazdan bej dönüşümüne) indüklendi. Ayrıca, in vitro Prdm16 aşırı ekspresyonu oksijen tüketimini arttırdı. Bu nedenle, bu protokol yağ dokusunda bej yağ gelişimi mekanizmalarını keşfetmek için çok yönlü bir SPH fare modeli sağlar.

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	<tbody>
		<tr>
			<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>
		</tr>
		<tr>
			<td>AAVpro 293T Cell Line</td>
			<td>Takarabio</td>
			<td>632273</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra Centrifugal Filter</td>
			<td>Merckmillipore</td>
			<td>UFC510008</td>
			<td>100 KDa</td>
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		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D1756</td>
			<td></td>
		</tr>
		<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>
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		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
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		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-25G</td>
			<td></td>
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		<tr>
			<td>Hexadimethrine bromide (Polybrene)</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<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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		<tr>
			<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>
		</tr>
		<tr>
			<td>pAAV-U6-gRNA-CBh-mCherry</td>
			<td>Addgene&#160;</td>
			<td>91947</td>
			<td></td>
		</tr>
		<tr>
			<td>pAdDeltaF6&#160;</td>
			<td>Addgene&#160;</td>
			<td>112867</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
	</tbody>
</table>

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

Kümelenmiş düzenli aralıklı kısa palindromik tekrarlar (CRISPR) teknolojisi biyolojide bir devrim yarattı ve yeni araçlar orijinal olarak tarif edilen gen düzenlemesinin çok ötesinde uygulandı. CRISPR aktivasyonu (CRISPRa) sistemi, endojen gen ekspresyonunu indüklemek için katalitik olarak inaktif Cas9 (dCas9) proteinini farklı transkripsiyon modülleri ile birleştirir. SunTag-p65-HSF1 (SPH), sinerjik aktivasyon mediatörlerinin (SAM) bileşenlerini SunTag aktivatörleri ile birleştiren yakın zamanda geliştirilmiş bir CRISPRa teknolojisidir. Bu sistem, özelleştirilmiş bir tek kılavuzlu RNA (sgRNA) tasarlayarak tek veya çoklu genlerin aşırı ekspresyonuna izin verir. Bu çalışmada, AdipoSPH adı verilen adipositlerde (adiponektin Cre soyundan) SPH eksprese eden koşullu bir fare oluşturmak için daha önce geliştirilmiş bir SPH faresi kullanılmıştır. Beyaz-bej yağ (kahverengileşme) fenotipini indüklemek için, endojen Prdm16 genini (kahverengi ve bej yağ gelişimi ile ilgili iyi kurulmuş bir transkripsiyon faktörü) hedef alan sgRNA taşıyan adeno ilişkili bir virüs (AAV), inguinal beyaz yağ dokusuna (iWAT) enjekte edildi. Bu fare modeli, endojen Prdm16 ekspresyonunu indükledi ve termojenik gen programını aktive etti. Ayrıca, in vitro SPH kaynaklı Prdm16 aşırı ekspresyonu, bej adipositlerin oksijen tüketimini arttırdı ve önceki bir Prdm16 transgenik fare modelinin sonuçlarını fenotopkopyaladı. Bu nedenle, bu protokol yağ dokusu biyolojisini araştırmak için çok yönlü, uygun maliyetli ve zaman etkili bir fare modelini tanımlar.

AdipoSPH fareleri, SPH ve Adipoq-Cre fare suşlarının üremesiyle geliştirilmiştir. Her iki fare suşu da hibrit bir C57BL6J-DBA / 2J arka planındaydı (ticari tedarikçiye göre; bkz. SPH fare soyu başlangıçta Zhou ve ark.14 tarafından tanımlanmıştır.
 AdipoSPH aracılı Prdm16 aşırı ekspresyonu yoluyla in vivo bej adiposit gelişimi Bu çalışmada açıklanan modelin in vivo bej 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

Bu protokol, bej yağ biyolojisini araştırmak için adeno ilişkili virüse (AAV) alternatif bir strateji olarak adipositlerde (AdipoSPH) CRISPR SunTag-p65-HSF1 (SPH) kullanımını sunmaktadır. Endojen Prdm16 genini hedef alan AAV taşıyan sgRNA'nın in vivo enjeksiyonu, bej yağ gelişimini indüklemek ve termojenik gen programını geliştirmek için yeterlidir.

CRISPR SunTag-p65-HSF1 Aktivasyon Sistemi Kullanılarak Bej Yağ Biyolojisi ve Metabolizmasının İncelenmesi

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

Bu protokol, SPH CRISPR Aktivasyon Sistemini, adipositlerde kazanç fonksiyonu testleri yapmak için geleneksel viral vektörlere alternatif bir strateji olarak sunmaktadır. Adino ilişkili bir virüs kullanarak özelleştirilmiş tek kılavuzlu RNA vererek, yağ dokusunun karmaşık hücresel ortamında tek veya çoklu adiposit endojen genlerinin aşırı ekspresyonuna izin verir. Bu protokolün zorlu adımları, tek kılavuz RNA'nın klonlanması ve adeno ilişkili virüsün yağ dokusuna üretilmesi ve enjekte edilmesi ile ilgilidir.Başlamak için, chopchop veya başka bir uygun alet kullanarak CRISPR aktivasyonu için tek kılavuzlu RNA veya sgRNA tasarlayın. Aşağıdaki parametreleri kullanarak PRDM16 genini hedefleyen sgRNA'yı tasarlayın:Bir CRISPR / Cas9 kullanarak ve aktivasyon olarak bir Mus kaslarında PRDM16 olarak hedefleyin. Vektör omurgası PAAVU6GRNACBHM kirazındaki Sac1 kısıtlama bölgesini eşleştirmek için sgRNA'ya çıkıntılar ekleyin.5'AGCT 3'ü dahil edin, burada n nükleotitlere karşılık gelir. Belirtilen aracı kullanarak 3'sgRNA ters kompleman dizisi elde edin. Daha sonra, tek sarmallı ücretsiz oligonükleotidlerin tavlanması için, her 5've 3' tek sarmallı oligonükleotidin bir mikrolitresi, bir mikrolitre T4 ligaz tamponu, 0.5 mikrolitre T4 polinükleotid kinaz ve 10 mikrolitrelik bir son reaksiyon hacmi için 6.5 mikrolitre su ekleyin.Ücretsiz tek sarmallı oligonükleotidleri, 30 dakika boyunca 37 santigrat derecede ve beş dakika boyunca 95 santigrat derecede bir termosiklet kullanarak tavlayın, ardından dakikada beş santigrat derece rampa düşürme oranı izleyin. Daha sonra tavlanmış sgRNA oligonükleotidlerin ligasyonu için, iki mikrolitre tavlanmış sgRNA Oligonükleotid, bir mikrolitre Sac1 enzimi, iki mikrolitre 10X T4 DNA ligaz tamponu, bir mikrolitre T4 DNA ligaz ve suya 10 mikrolitrelik bir son reaksiyon hacmine 25 nanogram plazmid PAAVU6GRNACBHM kirazı ekleyin. Bir termosiklet kullanarak, reaksiyon karışımını beş dakika boyunca 37 santigrat derecede 15 döngü ve beş dakika boyunca 25 santigrat derecede inkübe ederek ve ardından dört santigrat derecede tutarak ligasyon gerçekleştirin.Daha sonra, yetkili Escherichia coli DH10B hücrelerini, ligasyon ürününün dört mikrolitresi ile 42 santigrat derecede 45 saniye boyunca ısı şoku ile dönüştürün ve mililitre Ampisilin başına 10 mikrogram içeren bir agar plakasına yayın. PCR Master Mix'i kullanarak Koloni Polimeraz Zincir Reaksiyonu veya PCR ile dönüştürülmüş kolonileri onaylayın, koloni seçin ve beş mikrolitre Master Mix, 0.1 mikrolitre evrensel astar, 0.1 mikrolitre sgRNA ters astar ve beş mikrolitre su ile karıştırın. PCR'yi iki dakika boyunca 94 santigrat derecede ilk denatürasyon, ardından 20 saniye boyunca 94 santigrat derecede 35 döngü denatürasyon, 60 santigrat derecede 30 saniye tavlama, 30 saniye boyunca 72 santigrat derecede uzatma ve beş dakika boyunca 72 santigrat derecede son bir uzama adımı kullanarak çalıştırın.PCR'den sonra, DNA'yı 30 dakika boyunca 90 voltta 0.5X TAE Tamponunda agaroz jel elektroforezi kullanarak çözün. Universal astar kullanarak Sanger Dizilimi için pozitif örnekler gönderin. Daha sonra, üreticinin talimatlarını izleyerek bir plazmid saflaştırma kiti kullanarak plazmidi pozitif bir klondan arındırın.Anestezi uygulanan fareyi sırtüstü pozisyona yerleştirin ve kasık beyazı yağ dokusu veya tıraş makinesi ile IWAT enjeksiyonları için kalça eklemlerine yakın yanlarda küçük bir alanı tıraş edin. Beş dakika boyunca tüy dökücü krem ekleyin. Cilt yanıklarını önlemek için kalan kremi suyla çıkarın.Povidon iyot çözeltisini cilde temiz gazlı bez ve% 70 alkol ile uygulamak için üç alternatif tur kullanarak cildi dezenfekte edin. Daha sonra, eklemlerin proksimal bölgesinde sterilize edilmiş makasla bir ila iki santimetrelik bir kesi yapın ve yağ deposunu ortaya çıkarmak için forseps kullanarak cildi açık tutun. WAT her iki taraftaki cilde tutturulabilir, baştan arkadan ve testislere doğru uzatılabilir.Forseps kullanarak, enjeksiyonun doğru yerde ve derinlikte olduğundan emin olmak için yağ deposunu insizyondan yavaşça yukarı doğru çekin. Mikrolitre şırıngayı 2.5 mikrolitre adeno ilişkili virüs veya endojen PRDM16 genini hedef alan sgRNA içeren bir AAV ile doldurun. İğneyi IWAT'a 30-45 derecelik bir açıyla dikkatlice yerleştirin.Tüm IWAT yağ yastığını homojen bir şekilde enfekte etmek için enjeksiyonu dokunun farklı yerlerine beş kez tekrarlayın. Bilinç yeniden kazanılana kadar fareyi ısıtma yastığına yerleştirin. Tamamen iyileşene kadar hayvanı her 10 ila 15 dakikada bir izleyin.Hayvan bilincini yeniden kazandıktan sonra, doğrusal olması ve sıkıntı veya ağrı belirtisi olmaması gereken lokomotif profillemesini gözlemleyin. Adipo SPH fare IWAT'ından türetilen stromal vasküler hücreleri veya SVF'leri, bir ila iki saat boyunca tam DMEM ortamı içeren 6 Kuyucuklu bir plakaya bakın, ortamı aspire edin, PBS kullanarak kuyuyu iki kez yıkayın ve taze, eksiksiz bir ortamla değiştirin. Hücreler% 70-80 akıcılığa ulaşana kadar hücreleri 37 santigrat derece,% 5 karbondioksit ve% 95 nemde inkübe edin.0. Günde, hücreleri indüksiyon ortamı ile tedavi ederek farklılaşmayı indükleyin ve iki gün sonra indüksiyon ortamını bakım ortamı ile değiştirin. Yine iki gün sonra, dördüncü günde, bakım ortamını 2-3 gün boyunca taze bir bakım ortamıyla değiştirin. Preadipositler adipositlere tamamen farklılaşana kadar bakım ortamını her 48 saatte bir değiştirin.Farklılaşmış hücreler lipit damlacıkları ile yüklü gibi göründüğü için ışık mikroskobu kullanarak olgun adipositleri gözlemleyin. Hücreler %70-80 akıcılığa ulaşana kadar hücreleri 6 Kuyucuklu bir kültür plakası üzerinde tam ortamla büyüttükten sonra, AAV taşıyan sgRNA PRDM16'nın mikrolitresi başına 5.6 * 10 ^ 10 viral genomu iki mililitre tam ortam ve hekzadimetrin bromür ile karıştırın. Tam ortamı değiştirerek ve AAV içeren tam ortamı ekleyerek hücreleri dönüştürün.Dönüştürülen hücreleri 12 saat boyunca 37 santigrat derece,% 95 nem ve% 5 karbondioksitte inkübe edin. Hücreleri bölün ve hücre proliferasyonu ve bej adipositlere farklılaşma için tarif edildiği gibi görün. Adipo SPH farelerinin IWAT'ından alınan immünofloresan görüntüleri, hem kontrol hem de PRDM16 gruplarında dCas9 ekspresyonunu göstermiştir.SPH'ye bağlı PRDM16 ekspresyonu, çok oküler bej adipositlerin IWAT'a yaygın bir şekilde birikmesine açıkça neden olmuştur. Ayrıca, kantitatif PCR, PRDM16 grubunda PRDM16 ekspresyonunun ve termojenik gen programının kontrole kıyasla arttığını ortaya koydu. Benzer şekilde, in vitro gen ekspresyon analizi, PRDM16 aşırı ekspresyon grubundaki endojen PRDM16 geninin ve termojenik genlerin kontrole kıyasla artmış ekspresyonunu doğruladı.SPH kaynaklı PRDM16 ekspresyonu, kontrol hücrelerindekinden daha yüksek bazal ve maksimum oksijen tüketimi ile sonuçlandı. Önemli olarak, veriler PRDM16 grubunda, kontrol grubundakine kıyasla bağlanmamış solunumun arttığını göstermiştir. Adipo SPH modeli, adipositler içindeki çoklu genleri ve düzenleyici elementleri araştırmak için uygundur.Bu yöntem, bej yağ biyolojisinin daha derin bir şekilde anlaşılması olasılığını ortaya çıkarır.

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.

Elektrokimyasal Sensörler kullanarak Bakteriyel Algılama ve Tanıma

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

Biyomühendislik

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.

Kontrast-bilgisayarlı Perfüzyon-sabit Örneklerinin Tomografi kullanarak İnsan Kardiyak Venöz Sistemi anatomik yeniden düzenlemesi

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.

180 ° Kavisli Arter Testi Bölüm Model Tip IV Stent Başarısızlık Mansap İkincil Akış Yapıların Deneysel İncelenmesi

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.

Diyabetik Farenin Epididimal Yağ Tabağında Iskelelerin İskele Destekli Nakli

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.

Yaşayan Hücreleri Mühendise Sentetik Biyoloji kullanarak That Programlanabilir Malzemelerle Arayüzü

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.

Verimli üretimi ve besleyici-Alerjik IPSCs CRISPR-Cas9 sistemini kullanarak insan pankreas hücreleri düzenleme

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.

Perfkullanılmış Mammalin kalpleri transmural absorbance spektroskopisi kullanarak hücresel metabolizmayı Izleme için kalp içi yan ateş ışık kateter

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 düzenleme Saccharomyces cerevisiae ve Maya Pixel Art oluşturulması

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.

CRISPR Plazmidlerinin Yapımı ve Çift Luciferase Muhabir Sistemi Ile Memeli Hücrelerinde Nakavt Etkinliğinin Saptanması

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.

Biplanar Videoradyografi kullanarak 3D In-vivo Omuz Kinematik ölçümü

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