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

作者感谢Unicamp的Centro Multidisciplinar para Investigação Biológica na Área da Animais de Laboratório（Cemib）对AdipoSPH小鼠的产生，免疫代谢和细胞信号实验室以及国家应用于细胞生物学的光子学科学技术研究所（INFABIC）的所有实验支持。我们感谢圣保罗研究基金会（FAPESP）的财政支持：2019/15025-5;2020/09308-1;2020/14725-0;2021/11841-2.

动物研究是根据坎皮纳斯大学实验动物护理和使用指南（协议CEUA #5810-1/2021）进行的。
1. 分子克隆
<ol><li>单向导RNA（sgRNA）的设计<ol><li>使用CHOPCHOP（https://chopchop.cbu.uib.no/ 提供）或任何其他合适的工具设计用于CRISPR激活的sgRNA。使用以下参数设计靶向Prdm16基因的sgRNA：靶标：Prdm16;在：肌肉肌肉;使用：Crispr/Cas9;对于：激活。 注意：为每个感兴趣的区域设计sgRNA，...

<ol>
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CRISPR技术最有用的非编辑应用之一是通过使用CRISPRa系统激活内源性基因来询问基因功能6。SPH是一种强大的CRISPRa，最初被描述为通过靶向几个神经源性基因来诱导星形胶质细胞转化为活跃的神经元14。在这项研究中，AdipoSPH被证明是通过激活脂肪细胞中内源性Prdm16的表达来研究米色脂肪生物学的合适工具。SPH诱导的Prdm16在脂肪细胞中的过表达导致产热基因程序的...

米色（或卤状）脂肪细胞是解偶联表达蛋白 1 （UCP1） 和富含线粒体的脂肪细胞，位于白色脂肪组织 （WAT） 库中。米色脂肪来自脂肪细胞祖细胞或成熟白色脂肪细胞的亚群，以响应寒冷暴露和其他刺激1，2。米色脂肪细胞可以以UCP1依赖性或独立的方式将能量转化为热量3。除了产热功能如何，米色脂肪还可以通过其他方式改善代谢健康，例如脂肪因子的分泌和抗炎和抗纤维化活性。对小鼠和人类的研究表明，米色脂肪的诱导改善全身葡萄糖和脂质稳态3。然而，尽管近年来我们对米色脂肪生物学的了解发展迅速，但其大部分代谢益处和相关机制仍未完全了解。
簇状规则间隔短回文重复序列（CRISPR）首先在真核细胞中被描述为能够通过Cas9蛋白4，5的核酸酶活性在基因组的特定位点产生双链断裂（DSB）的工具。Cas9由合成的单向导RNA（sgRNA）引导，靶向特定的基因组区域，从而产生DNA DSB。除了使用核酸酶Cas9进行编辑外，CRISPR-Cas9技术还发展成为序列特异性基因调控工具6。催化无活性Cas9蛋白（dCas9）的发展和能够增强基因表达的转录模块的结合产生了CRISPR激活（CRISPRa）工具。已经出现了几种CRISPRa系统，例如VP647，8，协同激活介质（SAM）9，SunTag10，11，VPR 12，13和SunTag-p65-HSF1（SPH）14，它们结合了SAM和SunTag激活剂的组件。最近已经证明，与其他CRISPRa系统相比，使用SPH诱导的N2a神经母细胞和原代星形胶质细胞中神经源性基因的表达更高14，这表明SPH是一种有前途的CRISPRa工具。
在这里，我们利用先前开发的SPH小鼠14 ，使用脂联素Cre谱系（AdipoSPH）生成在脂肪细胞中特异性表达SPH的条件小鼠模型。使用携带靶向内源性Prdm16基因的gRNA的腺相关病毒（AAV），诱导腹股沟WAT（iWAT）的褐变（白色到米色转换）以增加产热基因程序的表达。此外， 体外 Prdm16过表达增加了耗氧量。因此，该协议提供了一种多功能的SPH小鼠模型，用于探索脂肪组织内米色脂肪发育的机制。

<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

成簇规则间隔短回文重复（CRISPR）技术引发了生物学的革命，最近的工具已经远远超出了最初描述的基因编辑。CRISPR活化（CRISPRa）系统将催化无活性的Cas9（dCas9）蛋白与不同的转录模块相结合，以诱导内源性基因表达。SunTag-p65-HSF1（SPH）是最近开发的一种CRISPRa技术，它将协同激活介质（SAM）的成分与SunTag激活剂相结合。该系统通过设计定制的单向导RNA（sgRNA）允许单个或多个基因的过表达。在这项研究中，先前开发的SPH小鼠用于在脂肪细胞（脂联素Cre谱系）中产生表达SPH的条件小鼠，称为AdipoSPH。为了诱导白色至米色脂肪（褐变）表型，将携带靶向内源性Prdm16基因（一种与棕色和米色脂肪发育相关的成熟转录因子）的sgRNA的腺相关病毒（AAV）注射到腹股沟白色脂肪组织（iWAT）中。该小鼠模型诱导内源性Prdm16的表达并激活产热基因程序。此外， 体外 SPH诱导的Prdm16过表达增强了米色脂肪细胞的耗氧量，表型复制了先前Prdm16转基因小鼠模型的结果。因此，该协议描述了一种用于研究脂肪组织生物学的多功能，经济高效且具有时间效率的小鼠模型。

AdipoSPH小鼠是通过培育SPH和Adipoq-Cre小鼠品系开发的。两种小鼠品系均处于杂交C57BL6J-DBA / 2J背景中（根据商业供应商;见 材料表）。SPH小鼠谱系最初由Zhou等人描述14。
通过 AdipoSPH介导的Prdm16过表达实现体内米色脂肪细胞发育 为了评估本研究中描述的模型在 体内发育米色脂肪细胞的能力，将携带靶向Prdm16基...

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

该协议提出了在脂肪细胞（AdipoSPH）中使用CRISPR SunTag-p65-HSF1（SPH）作为腺相关病毒（AAV）的替代策略来研究米色脂肪生物学。 体内 注射靶向内源性Prdm16基因的携带AAV的sgRNA足以诱导米色脂肪发育并增强产热基因程序。

使用CRISPR SunTag-p65-HSF1激活系统研究米色脂肪生物学和代谢

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

该协议将SPH CRISPR激活系统作为传统病毒载体的替代策略，以在脂肪细胞中进行增益功能测定。它通过使用腺相关病毒递送定制的单向导 RNA 来允许在脂肪组织的复杂细胞环境中过表达单个或多个脂肪细胞内源性基因。该协议的挑战性步骤与克隆单向导RNA以及将腺相关病毒的产生和注射到脂肪组织中有关。首先，使用chopchop或任何其他合适的工具设计用于CRISPR激活的单向导RNA或sgRNA。使用以下参数设计靶向PRDM16基因的sgRNA：使用CRISPR / Cas9靶向肌肉中的PRDM16和作为激活。向 sgRNA 添加突出部分以匹配载体骨架 PAAVU6GRNACBHM 樱桃中的 Sac1 限制性位点。包括5'AGCT 3'，其中n对应于核苷酸。使用指示的工具获得 3'sgRNA 反向补体序列。接下来，对于单链互补寡核苷酸的退火，加入每5'和3'单链寡核苷酸各1微升，T4连接酶缓冲液1微升，T4多核苷酸激酶0.5微升和6.5微升水，最终反应体积为10微升。使用热循环仪在 37 摄氏度下退火 30 分钟，在 95 摄氏度下退火 5 分钟，然后以每分钟 5 摄氏度的降温速率退火。然后，为了连接退火的sgRNA寡核苷酸，将25纳克质粒PAAVU6GRNACBHM樱桃加入到2微升退火的sgRNA寡核苷酸，1微升Sac1酶，2微升10X T4 DNA连接酶缓冲液，1微升T4 DNA连接酶和水中，最终反应体积为10微升。使用热循环仪，通过将反应混合物在 37 摄氏度下孵育 15 个循环 5 分钟，在 25 摄氏度下孵育 5 分钟，然后在 4 摄氏度下保持来进行连接。接下来，通过42摄氏度的热休克45秒，用4微升的连接产物转化感受态大肠杆菌DH10B细胞，并铺展在含有每毫升氨苄青霉素10微克的琼脂平板上。使用PCR预混液通过菌落聚合酶链反应或PCR确认转化菌落，挑选菌落并将其与5微升预混液，0.1微升通用引物，0.1微升sgRNA逆转引物和5微升水混合。使用在 94 摄氏度下初始变性两分钟运行 PCR，然后在 94 摄氏度下变性 35 个循环 20 秒，在 60 摄氏度下退火 30 秒，在 72 摄氏度下延伸 30 秒，最后伸长步骤在 72 摄氏度下持续 5 分钟。PCR后，使用琼脂糖凝胶电泳在0.5X TAE缓冲液中以90伏特分离30分钟。使用通用引物提交阳性样品进行桑格测序。接下来，按照制造商的说明使用质粒纯化试剂盒从阳性克隆中纯化质粒。将麻醉小鼠置于仰卧位，并在髋关节近端的侧面剃除一小块区域，用于腹股沟白色脂肪组织或用剃须刀注射IWAT。加入脱毛膏五分钟。用水去除残留的乳霜，以避免皮肤灼伤。用干净的纱布和70%酒精交替将聚维酮碘溶液涂抹在皮肤上，对皮肤进行三轮消毒。然后，在关节的近端区域用消毒剪刀做一个一到两厘米的切口，并使用镊子保持皮肤打开以露出脂肪库。WAT可以附着在两侧的皮肤上，从背部开始延伸到睾丸。使用镊子，通过切口轻轻向上拉脂肪库，以确保注射处于正确的位置和深度。用2.5微升腺相关病毒或含有靶向内源性PRDM16基因的sgRNA的AAV填充微升注射器。小心地将针头以 30-45 度角插入 IWAT。在组织的不同位置重复注射五次，以均匀地感染整个IWAT脂肪垫。将鼠标放在加热垫上，直到恢复意识。每 10 到 15 分钟监测一次动物，直到它完全康复。动物恢复意识后，观察机车轮廓，它应该是线性的，没有痛苦或疼痛的迹象。将源自adipo SPH小鼠IWAT的基质血管细胞或SVF放入含有完整DMEM培养基的6孔板中一到两个小时，吸出培养基，使用PBS洗涤孔两次，并用新鲜的完全培养基替换。将细胞在37摄氏度，5%二氧化碳和95%湿度下孵育，直到细胞达到70-80%汇合。在第0天，通过用诱导培养基处理细胞来诱导分化，两天后用维持培养基替换诱导培养基。两天后，在第四天，再次用新鲜的维护介质更换维护介质 2-3 天。每48小时更换一次维持培养基，直到前脂肪细胞完全分化为脂肪细胞。使用光学显微镜观察成熟的脂肪细胞，因为分化的细胞似乎装有脂滴。在带有完整培养基的6孔培养板上生长细胞直至细胞达到70-80%汇合度后，将每微升携带AAV的sgRNA PRDM16的5.6 * 10^10病毒基因组与2毫升完全培养基和六二甲基溴混合。通过更换完整培养基并加入含有AAV的完整培养基来转导细胞。将转导的细胞在37摄氏度95%湿度和5%二氧化碳下孵育12小时。按照描述的细胞分裂并观察细胞增殖和分化为米色脂肪细胞。来自脂肪SPH小鼠IWAT的免疫荧光图像显示了dCas9在对照组和PRDM16组中的表达。SPH诱导的PRDM16表达明显诱导多眼米色脂肪细胞广泛积累到IWAT中。此外，定量PCR显示，与对照组相比，PRDM16组PRDM16和产热基因程序的表达增加。同样，体外基因表达分析证实，与对照组相比，PRDM16过表达组内源性PRDM16基因和产热基因的表达增加。SPH诱导的PRDM16表达导致的基础和最大耗氧量高于对照细胞。重要的是，数据显示，与对照组相比，PRDM16组的无耦合呼吸增强。Adipo SPH模型适用于研究脂肪细胞内的多个基因和调控元件。这种方法为更深入地了解米色脂肪生物学开辟了可能性。

Research

JoVE Journal

Bioengineering

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Bacterial Detection & Identification Using Electrochemical Sensors

使用电化学传感器的细菌检测和鉴定

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

科研

生物工程

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

人类心脏静脉系统使用对比计算机断层扫描灌注固定的标本解剖重建

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

在180°弧形动脉试验段的二次流动结构下游的型号IV型支架失败的实验研究

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

支架支持胰岛移植糖尿病小鼠附睾脂肪垫

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

利用合成生物学工程师活细胞接口，具有可编程材料

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

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

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

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

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

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

CRISPR/Cas12a 多路复用基因组编辑糖母细胞和酵母像素艺术的创作

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

通过双路西法酶检测系统构建CRISPR质粒和检测哺乳动物细胞的挖空效率

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. ...

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

使用压力表谱对毛质生殖系统的双轴基底音和被动测试

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). ...