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

著者らは、AdipoSPHマウスの作製、免疫代謝および細胞シグナル伝達研究所、および細胞生物学に適用されるフォトニクスに関する国立科学技術研究所(INFABIC)のすべての実験的支援に対して、Centro Multidisciplinar para Investigação Biológica na Área da Ciência em Animais de Laboratório(Cemib)、Unicampから受けた支援に感謝する。サンパウロ研究財団(FAPESP)からの財政的支援に感謝します:2019 / 15025-5;2020/09308-1;2020/14725-0;2021/11841-2.

動物実験は、実験動物の世話と使用のためのカンピーナス大学ガイド(プロトコルCEUA #5810-1 / 2021)に従って実施されました。
1. 分子クローニング
<ol><li>シングルガイドRNA(sgRNA)の設計<ol><li>CRISPR活性化用のsgRNAは、https://chopchop.cbu.uib.no/ で入手可能なCHOPCHOPまたはその他の適切なツールを使用して設計します。以下のパラメータを使用して、Prdm16遺伝子を標的と?...

<ol>
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CRISPR技術の最も有用な非編集アプリケーションの1つは、CRISPRaシステムを使用した内因性遺伝子の活性化による遺伝子機能の調査です6。SPHは強力なCRISPRaであり、いくつかの神経原性遺伝子を標的とすることによって星状細胞の活性ニューロンへの変換を誘導することが最初に記載されていました14。この研究では、AdipoSPHは、脂肪細胞における内因性Prdm16?...

ベージュ(またはブライト)脂肪細胞は、白色脂肪組織(WAT)デポー内に存在する脱共役タンパク質1(UCP1)発現およびミトコンドリアに富む脂肪細胞です。ベージュ色の脂肪は、低温曝露および他の刺激に応答して脂肪細胞前駆細胞または成熟白色脂肪細胞のサブセットから出現する1,2。ベージュ色の脂肪細胞は、UCP1依存的または独立した方法でエネルギーを熱に変換することができます3。ベージュ色の脂肪は、その熱発生機能に関係なく、アディポカインの分泌や抗炎症および抗線維化活性などの他の手段によって代謝の健康を改善することもできます。マウスとヒトでの研究では、ベージュ脂肪の誘導が全身のグルコースと脂質の恒常性を改善することが示されています3。しかし、ベージュ脂肪生物学に関する私たちの知識は近年急速に進化していますが、その代謝の利点と関連するメカニズムのほとんどはまだ完全には理解されていません。
クラスター化された規則的に間隔を空けた短い回文反復(CRISPR)は、Cas9タンパク質4,5のヌクレアーゼ活性を介してゲノムの特定の部位に二本鎖切断(DSB)を生成できるツールとして真核細胞で最初に説明されました。Cas9は、合成シングルガイドRNA(sgRNA)によって誘導され、特定のゲノム領域を標的とし、DNA DSBにつながります。編集目的でヌクレアーゼCas9を使用することに加えて、CRISPR-Cas9技術は、配列特異的遺伝子調節ツール6として使用されるように進化した。触媒的に不活性なCas9タンパク質(dCas9)の開発と、遺伝子発現を増強することができる転写モジュールの会合により、CRISPR活性化(CRISPRa)ツールが生まれました。VP64 7,8、相乗的活性化メディエーター(SAM)9、SunTag10,11、VPR 12,13、SAMとSunTagアクチベーターのコンポーネントを組み合わせたSunTag-p65-HSF1(SPH)14など、いくつかのCRISPRaシステムが登場しました。最近、N2a神経芽細胞および初代星状細胞における神経原性遺伝子の誘導発現が、他のCRISPRaシステムと比較してSPHを使用して高いことが実証され14、SPHが有望なCRISPRaツールとして実証されています。
ここでは、先に開発したSPHマウス14 を利用して、アディポネクチンCre系統(AdipoSPH)を用いて脂肪細胞に特異的にSPHを発現する条件付きマウスモデルを作製した。内因性Prdm16遺伝子を標的とするgRNAを搭載したアデノ随伴ウイルス(AAV)を用いて、鼠径WAT(iWAT)の褐変(白からベージュへの変換)を誘導し、熱発生遺伝子プログラムの発現を増加させた。さらに、 インビトロ Prdm16過剰発現は酸素消費量を増加させた。したがって、このプロトコルは、脂肪組織内のベージュ脂肪発生のメカニズムを探索するための汎用性の高いSPHマウスモデルを提供します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-25G</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<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>
		<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

クラスター化された規則的に間隔を空けた短い回文反復(CRISPR)技術は生物学の革命を引き起こし、最近のツールは最初に記述された遺伝子編集をはるかに超えて適用されています。CRISPR活性化(CRISPRa)システムは、触媒的に不活性なCas9(dCas9)タンパク質と異なる転写モジュールを組み合わせて、内因性遺伝子発現を誘導します。SunTag-p65-HSF1(SPH)は、相乗的活性化メディエーター(SAM)の成分とSunTagアクチベーターを組み合わせた最近開発されたCRISPRa技術です。このシステムは、カスタマイズされたシングルガイドRNA(sgRNA)を設計することにより、単一または複数の遺伝子の過剰発現を可能にします。本研究では、以前に開発したSPHマウスを用いて、脂肪細胞(アディポネクチンCre系統)でSPHを発現するAdipoSPHという名前の条件付きマウスを作製しました。白からベージュの脂肪(褐変)表現型を誘導するために、内因性Prdm16遺伝子(褐色およびベージュ色の脂肪の発生に関連する確立された転写因子)を標的とするsgRNAを搭載したアデノ随伴ウイルス(AAV)を鼠径部白色脂肪組織(iWAT)に注入した。このマウスモデルは、内因性Prdm16の発現を誘導し、熱発生遺伝子プログラムを活性化しました。さらに、 インビトロ SPH誘導Prdm16過剰発現は、ベージュ脂肪細胞の酸素消費を増強し、以前のPrdm16トランスジェニックマウスモデルの結果を表現コピーした。したがって、このプロトコルは、脂肪組織生物学を調査するための、用途が広く、費用効果が高く、時間効率の高いマウスモデルを記述します。

AdipoSPHマウスは、SPHおよびAdipoq-Creマウス系統を繁殖させることによって開発された。両方のマウス系統は、ハイブリッドC57BL6J-DBA/2Jバックグラウンドにあった(商業サプライヤーによる; 材料表を参照)。SPHマウス系統は、もともとZhouらによって記載された14。
AdipoSPHを介したPrdm16過剰発現によるインビボベージ...

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

このプロトコルは、ベージュ脂肪生物学を調査するためのアデノ随伴ウイルス(AAV)の代替戦略として、脂肪細胞(AdipoSPH)におけるCRISPR SunTag-p65-HSF1(SPH)の使用を示しています。内因性Prdm16遺伝子を標的とするAAV担持sgRNAの インビボ 注射は、ベージュ脂肪の発生を誘導し、熱発生遺伝子プログラムを増強するのに十分である。

CRISPR SunTag-p65-HSF1活性化系を用いたベージュ脂肪の生態と代謝の検討

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

このプロトコルは、脂肪細胞でゲイン機能アッセイを実行するための従来のウイルスベクターの代替戦略としてSPH CRISPR活性化システムを提示します。アデノ随伴ウイルスを使用してカスタマイズされた単一ガイドRNAを送達することにより、脂肪組織の複雑な細胞環境内で単一または複数の脂肪細胞内因性遺伝子の過剰発現を可能にします。このプロトコルの困難なステップは、単一ガイドRNAのクローニングと、脂肪組織へのアデノ随伴ウイルスの産生と注入に関連しています。まず、チョップチョップまたはその他の適切なツールを使用して、CRISPR活性化用のシングルガイドRNAまたはsgRNAを設計します。以下のパラメータを使用してPRDM16遺伝子を標的とするsgRNAを設計します:CRISPR/Cas9を使用して筋肉筋のPRDM16として標的とし、活性化として。ベクター骨格PAAVU6GRNACBHMチェリーのSac1制限部位と一致するようにsgRNAにオーバーハングを追加します。5'AGCT 3'を含み、ここでnはヌクレオチドに対応する。示されたツールを使用して3'sgRNA逆補体配列を取得します。次に、一本鎖相補的オリゴヌクレオチドのアニーリングのために、各5'および3'一本鎖オリゴヌクレオチド1マイクロリットル、T4リガーゼ緩衝液1マイクロリットル、T4ポリヌクレオチドキナーゼ0.5マイクロリットル、および6.5マイクロリットルの水を加えて、最終反応容量10マイクロリットルとする。相補的な一本鎖オリゴヌクレオチドをサーモサイクラーを用いて摂氏37度で30分間、摂氏95度で5分間アニーリングした後、毎分摂氏5度のランプダウン率でアニーリングします。次に、アニーリングされたsgRNAオリゴヌクレオチドのライゲーションのために、25ナノグラムのプラスミドPAAVU6GRNACBHMチェリーを2マイクロリットルのアニーリングされたsgRNAオリゴヌクレオチド、1マイクロリットルのSac1酵素、2マイクロリットルの10X T4 DNAリガーゼバッファー、1マイクロリットルのT4 DNAリガーゼおよび水を最終反応容量10マイクロリットルに加える。サーモサイクラーを用いて、反応混合物を摂氏37度で5分間、25度で5分間インキュベートした後、摂氏4度で保持してライゲーションを行います。次に、コンピテント大腸菌DH10B細胞を4マイクロリットルのライゲーション産物で摂氏42度で45秒間のヒートショックで形質転換し、1ミリリットルあたり10マイクログラムのアンピシリンを含む寒天プレートに広げます。PCRマスターミックスを用いたコロニーポリメラーゼ連鎖反応またはPCRによって形質転換されたコロニーを確認し、コロニーをピックし、5マイクロリットルのマスターミックス、0.1マイクロリットルのユニバーサルプライマー、0.1マイクロリットルのsgRNAリバースプライマー、および5マイクロリットルの水と混合します。初期変性を摂氏94度で2分間、続いて摂氏94度で20秒間35サイクル変性し、摂氏60度で30秒間アニーリングし、摂氏72度で30秒間伸長し、最後の伸長ステップを摂氏72度で5分間使用してPCRを実行します。PCR後、0.5X TAEバッファー中でアガロースゲル電気泳動を使用し、90ボルトで30分間DNAを分離します。ユニバーサルプライマーを使用したサンガーシーケンシングの陽性サンプルを提出してください。次に、製造元の指示に従ってプラスミド精製キットを用いて陽性クローンからプラスミドを精製する。麻酔をかけたマウスを仰臥位に置き、股関節の近位の脇腹の小さな領域を剃り、鼠径部の白色脂肪組織またはシェーバーによるIWAT注射を行います。脱毛クリームを5分間加えます。皮膚のやけどを防ぐために、残りのクリームを水で取り除きます。清潔なガーゼと70%アルコールで皮膚にポビドンヨード溶液を塗布する3ラウンドを交互に使用して皮膚を消毒します。次に、関節の近位部に滅菌ハサミで1〜2センチの切開を行い、鉗子を使用して皮膚を開いたままにして脂肪蓄積を露出させます。WATは両側の皮膚に取り付けることができ、最初から背中から精巣に向かって伸ばすことができます。鉗子を使用して、脂肪デポーを切開部からゆっくりと上に引き上げ、注射が正しい位置と深さにあることを確認します。マイクロリットルのシリンジに、内因性PRDM16遺伝子を標的とするsgRNAを含む2.5マイクロリットルのアデノ随伴ウイルスまたはAAVを充填します。針をIWATに30〜45度の角度で慎重に挿入します。組織の異なる場所に注射を5回繰り返して、IWAT脂肪パッド全体に均一に感染します。意識が回復するまでマウスを加熱パッドに置きます。完全に回復するまで、10〜15分ごとに動物を監視します。動物が意識を取り戻した後、機関車のプロファイリングを観察してください、それは直線的であるべきであり、苦痛や痛みの兆候がないはずです。脂肪SPHマウスIWATに由来する間質血管細胞またはSVFを、完全なDMEM培地を含む6ウェルプレートに1〜2時間観察し、培地を吸引し、PBSを使用してウェルを2回洗浄し、新鮮な完全培地と交換します。細胞が70〜80%のコンフルエント性に達するまで、摂氏37度、二酸化炭素5%、湿度95%で細胞をインキュベートします。0日目に、細胞を誘導培地で処理して分化を誘導し、2日後に誘導培地を維持培地と交換する。再び2日後、4日目に、2〜3日間、メンテナンス培地を新しいメンテナンス培地と交換します。脂肪前駆細胞が脂肪細胞に完全に分化するまで、48時間ごとに維持培地を交換してください。分化した細胞に脂肪滴がロードされているように見えるので、光学顕微鏡を使用して成熟脂肪細胞を観察します。細胞が70〜80%コンフルエントに達するまで、完全培地を含む6ウェル培養プレート上で細胞を増殖させた後、AAVを運ぶsgRNA PRDM16のマイクロリットルあたり5.6 * 10 ^ 10のウイルスゲノムを2ミリリットルの完全培地および臭化ヘキサジメトリンと混合します。完全な培地を交換し、AAVを含む完全な培地を添加することにより、細胞を形質導入します。形質導入した細胞を摂氏37度、湿度95%、二酸化炭素5%で12時間インキュベートします。細胞増殖およびベージュ脂肪細胞への分化について説明したように細胞を分割して見る。アディポSPHマウスのIWATからの免疫蛍光画像は、対照群およびPRDM16群の両方においてdCas9の発現を実証した。SPH誘導PRDM16発現は、多眼ベージュ脂肪細胞のIWATへの広範な蓄積を明らかに誘導した。さらに、定量PCRにより、PRDM16群では対照と比較してPRDM16および熱発生遺伝子プログラムの発現が増加していることが明らかになりました。同様に、インビトロ遺伝子発現解析により、PRDM16過剰発現群における内因性PRDM16遺伝子および熱発生遺伝子の発現増加が対照と比較して確認された。SPHによって誘導されたPRDM16発現は、対照細胞よりも高い基礎および最大酸素消費量をもたらしました。重要なことに、データは、対照群と比較してPRDM16群の非共役呼吸の増加を示した。Adipo SPHモデルは、脂肪細胞内の複数の遺伝子や調節要素の調査に適しています。この方法は、ベージュ脂肪生物学のより深い理解の可能性を開きます。

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.

電気化学センサを用いた細菌検出＆識別

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

生物工学

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.

灌流固定標本のコントラストコンピュータ断層撮影を用いた人間の心臓静脈系の解剖学的再建

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°湾曲動脈テストセクションにおけるモデルIV型ステントの失敗の下流に二次流れ構造に関する実験的研究

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.

糖尿病マウスの精巣上体脂肪細胞における膵島支持骨格移植

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.

プログラム可能な材料とのインタフェースリビング細胞を操作するために合成生物学を使用して、

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.

効率的な生成および CRISPR Cas9 システムを用いた人間の膵臓細胞から Ips をフィーダー フリーの編集

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.

浸透哺乳動物の心臓のトランスムラル吸光分光法を用いた細胞代謝モニタリングのための心臓内側発射光カテーテル

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 サッカロマイセスセレビシエの多重ゲノム編集と酵母ピクセルアートの創造

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.

二重ルシファーゼレポーターシステムによるCRISPRプラスミドの構築と哺乳動物細胞におけるノックアウト効率の検出

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.

ビプラナービデオラジオグラフィーを用いた3Dインビボショルダーキネマティクスの測定

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