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Neurociência

基于增强子的表达构建体在小鼠 大脑中的 AAV部署

Published: March 31, 2022
doi:
10.3791/62650
, ,
1Department of Psychiatry and Behavioral Sciences,University of California, Davis, 2Department of Neurobiology, Physiology and Behavior,University of California, Davis

Summary

该协议描述了一种新型基于rAAV的瞬时增强子 – 报告基因测定。该测定可用于在小鼠 大脑中诱导 增强子驱动的体内表达。

Abstract

增强子是多种转录因子的结合平台，可驱动组织和细胞类型特异性基因的特定表达模式。评估非编码DNA和各种染色质状态的多种方法已被证明可用于预测基因组中增强子序列的存在，但验证这些序列的活性并找到它们活跃的器官和发育阶段是一个劳动密集型过程。腺相关病毒（AAV）载体的最新进展使转基因能够广泛递送到小鼠组织，从而能够在不需要转基因动物的情况下进行 体内 增强子测试。该协议显示了在最小启动子的控制下表达EGFP的报告构建体如何用于研究小鼠大脑中候选增强子序列的活动模式，该启动子本身不会驱动显着表达。将AAV包装的报告构建体输送到小鼠大脑并孵育1-4周，之后处死动物，并在显微镜下观察脑切片。EGFP出现在测试增强子足以启动基因表达的细胞中，确定增强子在大脑中活跃的位置和发育阶段。标准的克隆方法、低成本的AAV包装以及扩展的AAV血清型和 体内 递送和标准成像读数的方法使其成为研究大脑中基因表达如何调节的可访问方法。

Introduction

增强子是作为转录因子结合位点的基因组顺式调控元件，可以以时空特异性的方式驱动靶基因的表达1，2。它们在不同的细胞类型、组织和发育阶段中具有差异活性，并且可以是疾病风险相关基因组变异的底物3，4。因此，了解增强子功能动态的需求对于基因组学中转化和基础科学应用的进展至关重要。在计算机中，增强子活性的预测可以作为产生增强子能力假设的极好资源5，6。这种预测的增强子活性可能需要额外的验证和询问，以充分了解功能活性。增强子报告基因测定已被证明在从细胞到动物的各种系统中具有此目的的价值7，8，9。为了在灵活且具有成本效益的瞬时体内环境中扩展这些研究，该协议描述了使用基于体内AAV的方法来测试推定的增强子序列，以了解它们在出生后小鼠大脑中驱动异位报告基因表达的能力。这一系列方法可用于询问单个候选序列或平行文库筛选，并且与基础和转化研究相关。

该方法在单个质粒中将推定的增强子候选DNA序列与报告基因（此处为EGFP）结合，在最小启动子的控制下，单独不驱动显着表达。将质粒包装成重组AAV（rAAV）并注射到动物模型中。虽然这里的应用是大脑，但各种rAAV血清型能够感染不同的组织类型，因此这种方法可以扩展到其他系统10。一段时间后，可以收集大脑并检测报告基因的表达。与对照组相比，强表达表明测试的候选序列能够“增强”基因的表达（图1）。这种简单的设计提供了一种简单明了的方法来测试 大脑中体内 增强子活性的序列。

除了测试序列的增强子能力外，该方法还可以与确定细胞类型增强子活性的技术相结合。在确定差异增强子活性的基于序列的方法中，在DNA和RNA测序之前对细胞类型特异性标记进行分选可以使研究人员确定不同的细胞类型是否显示出差异增强子活性，如Gisselbrecht等人11所述。在基于成像的方法中，将图像与细胞类型特异性标记物共标记，可以检查表现出增强子驱动荧光的细胞是否也显示感兴趣的细胞类型标记物12，13，14，15，16。增强子报告基因测定能够直接测试增强子中与风险相关的等位基因变异对增强子能力的影响。在全基因组关联研究（GWAS）中确定的绝大多数风险位点位于基因组的非编码区域17。这些风险位点的功能注释研究表明，很大一部分可能充当增强子18，19，20。MPRA在体内部署可以测试这些与风险相关的变异，以增强大脑中的活性12，21。最后，不同时间点的交付和收集可以提供对增强子活跃的发育阶段的见解。

增强子-报告质粒设计多种多样，可以定制以适应实验目标。有几种用于增强子研究的最小启动子的选择，例如人β珠蛋白最小启动子22 和小鼠Hsp68最小启动子23。已知这些启动子会驱动低水平的表达，除非与增强子元件耦合来激活它们。相反，组成型启动子元件驱动转基因的强表达，可用于阳性对照或在稳健表达的背景下测试增强子功能。组成型启动子的常见选择包括CAG，一种衍生自鸡β-肌动蛋白启动子和巨细胞病毒即时早期增强子24的杂交启动子24，或人EF1α25。由于已知增强子26双向工作，因此增强子相对于最小启动子的方向和位置是灵活的（图2A）。传统的增强子-报告基因测定将增强子置于启动子的上游，并且在文库递送中，包括报告基因下游的条形码序列，以将测序读数与测试的增强子27相关联。然而，增强子也可以放置在报告基因的开放阅读框架中，并作为它们自己的条形码序列，就像在STARR-seq28中所做的那样。这里描述的方案利用STARR-seq测定设计，将候选增强子序列放入报告基因的3′ UTR中。虽然STARR-seq取向提供了更简化的克隆的好处，但与传统方法相比，它不太清楚，并且可能在构建体之间诱导可变的RNA稳定性。所描述的方法可以很容易地适应STARR-seq或常规取向，对克隆过程进行微小的改变，这些改动已在其他地方描述27，29。

可以采用不同的AAV递送方法来进一步定制该技术以适应实验目标（图2B）。直接颅内注射，在本协议中进一步描述，将高浓度的病毒直接输送到大脑30。这提供了以注射部位为中心的高转导效率，使其成为希望最大化组织区域内转导细胞密度的实验的绝佳技术。立体定向注射可以帮助标准化动物的注射部位，以实现可重复的局部转导。颅内注射在出生后早期动物中最直接。作为一种替代技术，全身注射可以使用具有能够穿过血脑屏障的血清型的AAV传递转基因31。尾静脉注射允许病毒在全身循环，从而可以在许多组织中广泛传递10。眶后注射是另一种全身注射技术，可将病毒从眼睛后面输送到眶后窦32。这为AAV从静脉系统到大脑提供了更直接的途径，导致大脑中转导细胞的浓度高于注射到更多外周血管中的浓度33。

这种技术的另一个灵活方面是读出方法。从广义上讲，选项可以描述为基于报告基因或基于测序（图2C）。将荧光报告基因（如GFP）掺入构建体的开放阅读框中，导致荧光蛋白在候选增强子驱动表达的任何转导细胞中的表达。标记和成像技术（如免疫组织化学）可实现信号放大。基于测序的读出技术涉及从组织收集的RNA中从递送的构建体中识别序列。通过量化最初递送的病毒DNA的数量，表达RNA与递送DNA的比较可用于确定测试的增强子序列能够在多大程度上驱动转基因的表达增加，例如，在大规模平行报告测定（MPRA）的背景下。MPRA提供了这些技术的强大扩展，可以同时测试多达数千种候选增强子的活性，并在基因组学研究中进行了广泛的描述12，27，34，35，36。通过批量而不是单独对候选增强子执行克隆、包装、递送和测序步骤，可以实现更高的通量筛选。

候选增强子选择提供了另一个灵活性的机会（图 2D）。例如，该测定可用于鉴定特定基因的增强子，确定感兴趣的非编码DNA区域的功能，或确定增强子活跃的特定细胞类型或发育阶段 – 所有这些都服务于基础科学和疾病研究的目标。通常，候选增强子 的选择是由增强 子活性的计算机预测驱动的。通常， 计算机 预测包括组蛋白修饰的 ChIP-seq，这些修饰指示可能的增强子，例如 H3K27ac37 和染色质可及性映射38。最后，一个不断增长的研究领域是对合成设计的增强子元件进行基于功能的筛选，从而能够研究增强子序列如何指导功能39 和设计具有特定性质的增强子40。

Protocol

该协议已获得加州大学戴维斯分校机构动物护理和使用委员会（协议#22339）和加州大学戴维斯分校机构生物安全委员会（BUA-R1903）的批准。该协议已在出生后第0-1天在两性的C57BL / 6J小鼠上进行了测试。 1.将增强子候选序列克隆到AAV载体质粒中。 注意：给出了代表性的方案，但克隆策略具有高度的灵活性。 选择或设计报告器构造。在?…

Representative Results

使用这些方法，测试了基因CACNA1C19，49，50的精神风险相关第三内含子中的915 bp序列在出生后小鼠大脑中的增强子活性。该序列是在以精神和神经风险SNPs 12为中心的345个候选增强子序列的MPRA中发现的，这里将表征实验描述为一般示例。在Hsp68最小启动子和单个候选增强子序列的控制下，在P0上注射AAV9构?…

Discussion

该协议描述了一种基于rAAV的方法，用于在出生后小鼠大脑中部署增强子驱动的转基因。在这个通用的方案中，将候选增强子、最小启动子、报告基因和可选的条形码序列克隆到 AAV 质粒骨架中。这些实验可以用单个候选增强子序列或多个序列并行完成。质粒被包装成rAAV并输送到出生后的小鼠大脑。在一段时间允许病毒转导后，收集大脑并进行成像以进行报告基因表达。包括一种组成型表达的报告…

Declarações

The authors have nothing to disclose.

Acknowledgements

测序在加州大学戴维斯分校DNA技术核心进行。我们感谢加州大学戴维斯分校林田实验室对rAAV包装的培训，并慷慨地赠送给我们AAV助手和代表/帽质粒。这项工作得到了NIH / NIGMS R35GM119831的支持。

Materials

10x Citrate Buffer Sigma-Aldrich C9999-1000ML
5'-gatcactctcggcatggac-3' Integrated DNA Technologies N/A: Custom designed Forward primer for verifying clones after transformation. These primers are specific to the vector used and were designed for the specific vector used in our experiments.
5'-gatggctggcaactagaagg-3' Integrated DNA Technologies N/A: Custom designed Reverse primer for verifying clones after transformation. These primers are specific to the vector used and were designed for the specific vector used in our experiments.
Agarose VWR VWRVN605-500G
Aspirator tube assemblies Sigma-Aldrich A5177-5EA for mouth-driven delivery of rAAV
Bacteriological petri dishes Thermo Fisher Scientific 08-757-100D
Carbenicillin Sigma-Aldrich C1389-5G
Chicken IgY anti-GFP Thermo Fisher Scientific A10262
Confocal microscope Zeiss LSM900 The images were taken on the LSM800 model, but Zeiss launched the LSM900 model in recent years to replace LSM800.
Conical centrifuge tubes 15 mL Thermo Fisher Scientific 12-565-269
Cryomolds Thermo Fisher Scientific NC9806558 These molds are suitable for P28 mouse brain. Other sizes may be more suitable for larger or smaller tissues.
DAPI Sigma-Aldrich D9542-10MG
Dissecting scissors, 4.5" VWR 82027-578
Donkey anti-chicken AlexaFlour-488 Jackson ImmunoResearch 703-545-155
Dulbecco's PBS 1x Thermo Fisher Scientific MT21031CV
Eppendorf Microcentrifuge tubes 2.0 mL Thermo Fisher Scientific 22431048
Falcon round-bottom tubes 14 mL Thermo Fisher Scientific 352059
Fast Green dye Grainger F0099-1G
Fine detail paint brush set Artbrush Tower B014GWCLFO
Gibson Assembly Master Mix NEB E2611S
Glass capillary tubes Drummond Scientific Company 5-000-2005
HiSpeed Plasmid Maxi Kit QIAGEN 12663 Commercial plasmid maxi prep kit
HyClone HyPure Molecular Biology Grade Water VWR SH30538.03
IV butterfly infusion set with 12" tubing and 25G needle Thermo Fisher Scientific 26708
Kimwipes Kimberly Clark 34155 Lint-free wipe
LB Agar Thermo Fisher Scientific BP1425-500 LB agar pre-mix for selective media
McPherson Vannas iris scissor Integra LifeSciences 360-215
Mineral oil Sigma Life Science 69794-500ML
NEB Stable Competent E. coli NEB C3040I
NucleoSpin Gel and PCR Clean-Up Takara 740609.5 Kit for enzymatic reaction cleanup and gel extraction
OCT medium VWR 25608-930
Orbital shaker Cole Parmer 60-100
Paraformaldehyde Sigma-Aldrich 158127-500G
PCR strip tubes 0.2 mL VWR 490003-692
Peristaltic pump Gilson F155005
Phosphate buffered saline (PBS) 10x Thermo Fisher Scientific 70011044
Phusion Hot Start II High Fidelity DNA Polymerase Thermo Fisher Scientific F549L
Powdered milk Sunny Select
ProLong Gold Antifade Mountant Thermo Fisher Scientific P36934
QIAquick PCR Purification Kit QIAGEN 28106
rCutSmart Buffer NEB B6004S Buffer for restriction digest with PacI, AscI, and XmaI
Restriction enzyme: AscI NEB R0558L
Restriction enzyme: PacI NEB R0547L
Restriction enzyme: XmaI NEB R0180L
SOC outgrowth medium NEB B0920S Recovery medium after transformation
Sucrose (RNase/DNase free) Millipore Sigma 033522.5KG
TAE buffer Apex 20-194
Transfer tubing Gilson F1179941 For peristaltic pump
Triton X100 Sigma-Aldrich X100-100ML
Wizard Plus SV Minipreps DNA Purification System Thermo Fisher Scientific A1460 Plasmid mini prep kit
DOWNLOAD MATERIALS LIST

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AAVEnhancerExpressionReporterConstructCloningPCRGibson CloningPlasmidVirus TiterIn VivoMouse Brain
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Warren, T. L., Lambert, J. T., Nord, A. S. AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain. J. Vis. Exp. (181), e62650, doi:10.3791/62650 (2022).
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