Name: generation of brown fat-specific knockout mice using a combined cre-loxp, crispr-cas9, and adeno-associated virus single-guide rna system
Uploaded: 2023-03-24T13:00:00
Description: Watch this Scientific Journal Video about Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System at JoVE.com

这项工作得到了美国国立卫生研究院（NIH）拨款R01DK122808，R01DK102898和R01DK132469（给Y.-H.T.）以及P30DK036836（给乔斯林糖尿病中心的糖尿病研究中心）的部分支持。T.T.得到了SUNSTAR研究奖学金（日本Sunstar基金会Hiroo Kaneda奖学金）和美国心脏协会赠款903968的支持。Y. Z.得到了Charles A. King Trust Fellowship的支持。我们感谢Sean D. Kodani友好地校对手稿。

所有动物实验和护理程序都得到了乔斯林糖尿病中心机构动物护理和使用委员会的批准。
1. 筛选培养细胞中的有效sgRNA
注意：为了使测定具有成本效益，在将sgRNA包装成AAV颗粒之前，我们建议通过基于慢病毒的系统 在 培养细胞中测试不同的sgRNA，用于Cas9 / sgRNA表达（图1），并选择为 体内 实验提供最高敲低?...

<ol>
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现有已发表的AAV介导的基因递送至肩胛间BAT的方法不包含详细的图片和视频，描述将AAV直接注射到BAT中的手术技术和方法。在大多数已发表的方法33，34中，AAV被注射到肩胛间BAT周围的脂肪组织中，而不是BAT本身。因此，该协议中有几个关键步骤决定了研究的成功。这些包括sgRNA的设计，AAV包装和浓度，BAT特异性Cas9小鼠的产生以及外科手术。完成体内...

肥胖症在世界范围内以显着的速度增加，导致广泛的代谢疾病1，2，3。脂肪组织是这些病理的关键。存在的两种功能不同的脂肪组织类型是储存多余卡路里的白色脂肪组织（WAT）和棕色脂肪组织（BAT）及其相关的米色/盐渍脂肪，它们消耗能量以进行产热。虽然BAT因其耗能功能而得到认可，但它也通过产生调节远端器官代谢的生物活性分子而具有内分泌功能4，5。对啮齿动物的大量研究表明，增加棕色或米色脂肪的数量或活性会导致能量消耗增加和胰岛素敏感性提高。在人类中，可检测到BAT的人心脏代谢疾病的患病率显着降低6。因此，BAT对肥胖相关的代谢后遗症具有极好的治疗潜力7，8，9。
为了研究BAT发育和功能的生理学和病理生理学，并阐明这些过程中涉及的分子机制，BAT特异性转基因小鼠模型是选择的方法10。Cre-LoxP重组系统是通过编辑小鼠基因组来产生条件敲除小鼠的最常用手段。该系统能够以组织/细胞特异性方式修饰（过表达或敲除）感兴趣的基因11。它还可用于通过表达选择性荧光报告基因来标记特定细胞类型。
最近，通过结合CRISPR-Cas9技术和腺相关病毒（AAV）单向导RNA（sgRNA）系统12，进一步发展了Cre-LoxP系统方法。CRISPR-Cas9系统是一种特异性且高效的基因编辑工具，用于修饰，调节或靶向基因组的精确区域13。基于CRISPR-Cas9的基因组编辑允许对基因组位点进行快速遗传操作，因为它不需要与基因靶向载体进行同源重组。结合的Cre-LoxP，CRISPR-Cas9和AAV-sgRNA技术使研究人员能够通过研究目标基因在组织/细胞中所需时间的作用来更精确地了解基因功能。此外，如果使用诱导的Cre系，这些组合技术减少了产生转基因小鼠所需的时间和精力，并允许对CRISPR-Cas9活性进行时间控制，以便在小鼠中进行诱导基因组编辑14。
AAV载体是安全有效的体内基因递送系统。然而，靶向脂肪组织的AAV落后于其他组织的应用，例如大脑，心脏，肝脏和肌肉15。由于天然血清型载体的转导效率和嗜性相对较低，AAV引导的基因递送到脂肪组织仍然具有挑战性15。在过去的5年中，我们和其他人已经成功地建立了有效的微创方法，将AAV引导的基因递送到脂肪组织中，并创建了小鼠模型，使我们能够了解参与调节BAT功能的基因16，17，18，19。例如，通过使用AAV8将编码12-脂氧合酶（12-LOX）的靶向Alox12的sgRNA递送到Ucp1-Cre / Cas9小鼠的BAT中，我们发现活化的BAT产生12-LOX代谢物，即12-羟基二十碳五烯酸（12-HEPE）和13R，14S-二羟基二十二碳六烯酸（maresin 2），分别调节葡萄糖代谢和解决肥胖相关炎症16，17.在这里，我们提供了关于技术程序的分步方案，特别是将AAV-sgRNA直接显微注射到BAT叶中的手术，以使用组合的Cre-LoxP，CRISPR-Cas9和AAV-sgRNA系统产生BAT特异性敲除小鼠。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals, Peptides and Recombinant Proteins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OPTI-MEM serum-free medium</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene Infection / Transfection Reagent</td>
			<td>Sigma</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>TransIT-LT1 Transfection Reagent</td>
			<td>Mirus</td>
			<td>MIR 2300</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant DNA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>lentiCRISPR v2 vector&#160;</td>
			<td>Addgene</td>
			<td>52961</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-U6-BbsI-gRNA-CB-EmGFP</td>
			<td>Addgene</td>
			<td>89060</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G envelope plasmid</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2 packaging plasmid</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental Models: Cell Lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK-293 cells</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>WT-1 cells</td>
			<td>Developed in Tseng lab</td>
			<td>PMID: 14966273</td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental Models: Organisms/Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cas9 knockin mice</td>
			<td>Jackson Laboratories</td>
			<td>24857</td>
			<td></td>
		</tr>
		<tr>
			<td>Ucp1-CRE mice</td>
			<td>Jackson Laboratories</td>
			<td>24670</td>
			<td></td>
		</tr>
		<tr>
			<td>Others</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Banamine</td>
			<td>Patterson</td>
			<td>07-859-1323</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>Model 710 RN Syringe</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Fisher Scientific</td>
			<td>H325-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Hamilton</td>
			<td>32 gauge, small hub RN needle, needle point style 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture 5-0 Unify PCL25 P-3 Undyed 18 inch Monofilament 12/Bx</td>
			<td>Henry Schein</td>
			<td>1273504</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture 5-0 Unify Silk P-3 Black 18 inch 12/Bx</td>
			<td>Henry Schein</td>
			<td>1294522</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of brown fat-specific knockout mice using a combined cre-loxp, crispr-cas9, and adeno-associated virus single-guide rna system

棕色脂肪组织（BAT）是一种专门用于能量耗散的脂肪库，也可以通过分泌生物活性分子 作为 内分泌器官。创建BAT特异性敲除小鼠是了解目的基因对BAT介导的能量调节的贡献的最流行方法之一。利用Cre-LoxP系统的传统基因靶向策略一直是产生组织特异性敲除小鼠的主要方法。但是，这种方法既耗时又乏味。在这里，我们描述了一种使用组合的Cre-LoxP，CRISPR-Cas9和腺相关病毒（AAV）单向导RNA（sgRNA）系统快速有效地敲除BAT中目的基因的方案。肩胛间蝙蝠位于肌肉之间的深层。因此，必须对BAT进行曝光，以便将AAV精确地直接注入视野内的BAT。适当的手术处理对于防止交感神经和血管受损至关重要，例如连接到BAT的Sultzer静脉。为了尽量减少组织损伤，迫切需要了解BAT的三维解剖位置以及技术步骤中所需的手术技能。该协议重点介绍了关键技术程序，包括靶向目的基因的sgRNA的设计，AAV-sgRNA颗粒的制备，以及将AAV直接显微注射到两个BAT叶中以产生BAT特异性敲除小鼠的手术，可广泛应用于研究BAT中基因的生物学功能。

在上述程序中，AAV-sgRNA精确递送到BAT对于协议的成功至关重要。为了最大限度地提高AAV-sgRNA的效果并最大限度地减少手术过程中的组织损伤，了解BAT的三维（3D）解剖位置至关重要。如图3E所示，在不暴露组织的情况下很难确定BAT的精确位置。然而，过量的BAT暴露可能会损害组织（图3H，I）;因此，应执行该程序以在有限的解剖空间内注射AAV...

Watch this Scientific Journal Video about Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System at JoVE.com

Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System

在该协议中，我们描述了利用组合的Cre-LoxP，CRISPR-Cas9和腺相关病毒（AAV）单向导RNA（sgRNA）系统生成棕色脂肪组织（BAT）特异性敲除小鼠的技术程序。所描述的步骤包括sgRNA的设计，AAV-sgRNA颗粒的制备以及将AAV显微注射到BAT叶中。

使用组合的 Cre-LoxP、CRISPR-Cas9 和腺相关病毒单向导 RNA 系统生成棕色脂肪特异性敲除小鼠

Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive ...

该协议使用Cre-LoxP，CRISPR-Cas9和腺相关病毒单向导RNA系统生成棕色脂肪特异性敲除小鼠。它包括一种手术技术，可以安全地将AAV注入棕色脂肪中。该技术的主要优点是它可以快速产生组织特异性敲除，而无需选择性地繁殖小鼠。如果根据靶器官修改注射AAV的外科手术，则进行组织特异性敲除或敲除的方法可以适用于其他组织。首先，将镇痛药皮下施用到麻醉的UCP1 Cre Cas9小鼠中，以尽量减少和预防术后疼痛和痛苦。然后使用剃须刀在其肩胛间区域剃除皮毛，并用至少三轮交替使用碘基或氯己定基磨砂膏对手术区域进行消毒，然后用 70% 乙醇。使用手术刀，在肩胛骨之间的棕色脂肪组织上方的皮肤上切开一个两厘米的切口，以暴露肩胛间脂肪。做一个切口后，在脂肪组织和肌肉的远端区域之间的边界上切割脂肪组织。然后用惯用手的手术剪刀，将脂肪组织钝地垂直剥开，露出棕色脂肪组织，同时用非惯用手捏住脂肪组织，包括棕色脂肪组织。使用苏尔寿静脉作为标志来指示精确的棕色脂肪组织位置。接下来，对于每只小鼠，使用连接到100微升汉密尔顿注射器的锋利32号针头将20微升腺相关病毒或AAV缓慢注射到两个棕色脂肪组织的每个叶中。确认成功注射，表明在AAV给药期间注射部位没有泄漏。确保稳态后，将暴露的脂肪组织放回正常位置，使用5-0可吸收单丝线缝合脂肪组织和肌肉的边缘。最后，用 5-0 涂层 VICRYL 未染色的编织线缝合开放的皮肤。如果不暴露棕色脂肪组织，就无法确定其精确位置，但是，过度暴露对组织的损害。可能导致注射失败的潜在缺陷包括将AAV溶液注射到不正确的区域，针头穿透组织以及由于AAV溶液体积过大而导致组织膨胀。将AAV单向导RNA精确递送到棕色脂肪组织对于该方案的成功至关重要。暴露棕色脂肪组织时成功的关键是手术剪刀技术，包括剪刀的方向和深度，以及汉密尔顿注射器的位置。该技术具有产生负荷的组织特异性敲除动物，以了解棕色脂肪相关能量代谢的分子调节因子。

Research

JoVE Journal

Biology

Visualizing RNA Localization in Xenopus Oocytes

Visualizing RNA Localization in Xenopus Oocytes

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

科研

生物学

Substrate Generation for Endonucleases of CRISPR/Cas Systems

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

基因缺失在通过CRISPR / Cas9哺乳动物细胞系生成

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

AAV 滴定定量斑点印迹法及其在腺相关病毒组装活化蛋白功能评价中的应用

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

线虫的核苷酸和蛋白质提取联合

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

准备和注射 Culex 蚊子的胚胎，使用CRISPR/Cas9产生空突变

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

使用CRISPR/Cas9的人类B细胞基因组工程

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

使用杆状病毒-昆虫细胞培养系统生产和纯化腺相关病毒（AAV）2载体的工艺开发

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...