JoVE Journal > Bioengineering
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Abstract
Introduction
Protocol
Results
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Bioengineering

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

Published: July 06, 2021
doi:
10.3791/62109
, , ,
1Biomedical Engineering Department,Stony Brook University, 2Laufer Center for Physical and Quantitative Biology,Stony Brook University, 3Stony Brook Medical Scientist Training Program

Summary

可靠地控制光响应性哺乳动物细胞需要光遗传学方法的标准化。为了实现这一目标，本研究概述了基因电路构建，细胞工程，光遗传学设备操作和验证分析的管道，以标准化使用负反馈光遗传学基因电路作为案例研究的光诱导基因表达研究。

Abstract

无论使用何种方法，哺乳动物细胞中可靠的基因表达控制都需要具有高折叠变化、低噪声和确定的输入到输出传递函数的工具。为了实现这一目标，光遗传学基因表达系统在过去十年中因哺乳动物细胞中蛋白质水平的时空控制而受到广泛关注。然而，大多数控制光诱导基因表达的现有电路在结构上各不相同，由质粒表达，并利用可变光遗传学设备，因此需要探索稳定细胞系中光遗传学成分的表征和标准化。在这里，该研究提供了可靠的基因电路构建，整合和表征的实验管道，用于控制哺乳动物细胞中的光诱导基因表达，以负反馈光遗传学电路为例。这些方案还说明了标准化光遗传学设备和光照制度如何可靠地揭示基因电路特征，如基因表达噪声和蛋白质表达幅度。最后，本文可能适用于希望采用这种技术的光遗传学不熟悉的实验室。这里描述的管道应该适用于哺乳动物细胞中的其他光遗传学回路，从而可以在哺乳动物细胞的转录，蛋白质组学和最终表型水平上更可靠，更详细地表征和控制基因表达。

Introduction

与其他工程学科类似，合成生物学旨在标准化实验方案，允许使用具有高度可重复功能的工具来探索与生物系统相关的问题1，2。合成生物学中已经建立了许多控制系统的一个领域是基因表达调控领域3，4。基因表达控制可以靶向蛋白质水平和变异性（噪声或变异系数，CV = σ/μ，作为平均值的标准偏差测量），这是关键的细胞特征，因为它们在生理和病理细胞状态中的作用5，6，7，8。许多可以控制蛋白质水平和噪声的合成系统4，9，10，11，12 已经过设计，为跨工具标准化协议创造了机会。

最近出现的一套可以控制基因网络的新工具是光遗传学，能够使用光来控制基因表达13，14，15，16，17。与它们的化学前身类似，光遗传学基因回路可以引入任何细胞类型，从细菌到哺乳动物，允许表达任何感兴趣的下游基因18，19。然而，由于新型光遗传学工具的快速产生，许多系统在遗传电路结构、表达机制（例如，基于质粒与病毒整合）和供光控制设备方面存在差异11，16，20，21，22，23，24，25.因此，这为光遗传学特征的标准化留下了空间，例如基因电路构建和优化，系统利用方法（例如，积分与瞬态表达），用于诱导的实验工具以及结果分析。

为了在哺乳动物细胞中标准化光遗传学方案方面取得进展，该协议描述了一种实验管道，以使用集成到HEK293细胞（人类胚胎肾细胞系）中的负反馈（NF）基因电路来设计哺乳动物细胞中的光遗传学系统为例。NF是证明标准化的理想系统，因为它在本质上非常丰富26，27，28 ，允许调整蛋白质水平并实现噪声最小化。简而言之，NF允许通过抑制器精确控制基因表达，从而足够快地减少其自身表达，从而限制远离稳定状态的任何变化。稳态可以通过诱导剂来改变，该诱导剂使抑制剂失活或消除抑制剂，以允许更多的蛋白质产生，直到每个诱导剂浓度达到新的稳态。最近，一种工程NF光遗传学系统被创建出来，该系统可以产生基因表达的宽动态响应，保持低噪声，并对光刺激做出反应，从而具有空间基因表达控制的潜力11。这些工具被称为光诱导调节器（LITers），其灵感来自早期的系统，该系统允许在活细胞中进行基因表达控制4，10，29，30 ，并稳定地整合到人类细胞系中以确保长期的基因表达控制。

在这里，以LITer为例，概述了一种方案，用于创建光响应基因电路，使用光板装置（LPA，一种光遗传学诱导硬件）诱导基因表达31，并分析工程化的光遗传学可控细胞系对定制光刺激的反应。该协议允许用户将LITer工具用于他们希望探索的任何功能基因。它还可以通过集成下面概述的方法和光遗传学设备，适用于具有不同电路架构（例如，正反馈、负调节等）的其他光遗传学系统。与其他合成生物学方案类似，此处概述的视频记录和光遗传学方案可以应用于不同领域的单细胞研究，包括但不限于癌症生物学，胚胎发育和组织分化。

Protocol

1. 基因电路设计 选择遗传成分以组合成单个基因回路/质粒（例如，哺乳动物DNA整合序列基序32，光响应元件33或功能基因34）。 使用任何基因工程和/或分子克隆软件，存储DNA序列以供以后使用和参考，注释每个序列，并检查所有必要的特征（例如，START密码子，调控或翻译序列）35。 根据整体基因电路?…

Representative Results

本文中的基因电路组装和稳定细胞系生成基于含有转录活性，单个稳定FRT位点的商业改良HEK-293细胞（图1）。基因回路被构建到质粒内具有FRT位点的载体中，允许Flp-FRT整合到HEK-293细胞基因组中。这种方法不仅限于Flp-In细胞，因为FRT位点可以使用DNA编辑技术（如CRISPR / Cas950）添加到基因组中任何感兴趣的细胞系中。 一旦构建并验证了正?…

Discussion

本文的读者可以深入了解表征光遗传学基因回路（以及其他基因表达系统）至关重要的步骤，包括1）基因回路的设计，构建和验证;2）用于将基因电路引入稳定细胞系的细胞工程（例如，Flp-FRT重组）;3）使用基于光的平台（如LPA）诱导工程电池;4）通过荧光显微镜对光诱导测定进行初始表征;5）通过各种测定进行最终的基因表达表征，包括流式细胞术，定量实时荧光定量PCR（qRT-PCR）或免疫荧光测?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们要感谢Balázsi实验室的意见和建议，Karl P. Gerhardt博士和Jeffrey J. Tabor博士帮助我们构建了第一个LPA，以及Wilfried Weber博士分享了LOV2-degron质粒。这项工作得到了美国国立卫生研究院[R35 GM122561和T32 GM008444]的支持;劳弗物理与定量生物学中心;以及国防科学与工程研究生（NDSEG）奖学金。开放获取费用的资金：NIH [R35 GM122561]。

作者贡献：M.T.G.和G.B.构思了这个项目。M.T.G.，D.C.和L.G.进行了实验。M.T.G.、D.C.、L.G.和G.B分析了数据并准备了手稿。G.B.和M.T.G.监督了这个项目。

Materials

0.2 mL PCR tubes Eppendorf 951010006 reagent for carrying out PCR
0.25% Trypsin EDTA 1X Thermo Fisher Scientific MT25053CI reagent for splitting & harvesting mammalian cells
0.5-10 μL Adjustable Volume Pipette Eppendorf 3123000020 tool used for pipetting reactions
100-1000 μL Adjustable Volume Pipette Eppendorf 3123000039 tool used for pipetting reactions
20-200 μL Adjustable Volume Pipette Eppendorf 3123000055 tool used for pipetting reactions
2-20 μL Adjustable Volume Pipette Eppendorf 3123000039 tool used for pipetting reactions
5 mL Polystyene Round-Bottom Tube w/ Cell Strainer Cap Corning 352235 reagent for flow cytometry
5702R Centrifuge, with 4 x 100 Rotor, 15 and 50 mL Adapters, 120 V Eppendorf 22628113 equipment for mammalian culture work
Agarose Denville Scientific GR140-500 reagent for gel electrophoresis
Aluminum Foils for 96-well Plates VWR® 60941-126 tool used for covering plates in light-induction experiments
Ampicillin Sigma Aldrich A9518-5G reagent for selecting bacteria with correct plasmid
Analog vortex mixer Thermo Fisher Scientific 02215365PR tool for carrying PCR, transformation, or gel extraction reactions
Bacto Dehydrated Agar Fisher Scientific DF0140010 reagent for growing bacteria
BD LSRAria BD 656700 tool for sorting engineered cell lines into monoclonal populations
BD LSRFortessa BD 649225 tool for characterizing engineered cell lines
BSA, Bovine Serum Albumine Government Scientific Source SIGA4919-1G reagent for IF incubation buffer
Cell Culture Plate 12-well, Clear, flat-bottom w/lid, polystyrene, non-pyrogenic, standard-TC Corning 353043 plate used for growing monoclonal cells
Centrifuge VWR 22628113 instrument for mammalian cell culture
Chemical fume hood N/A N/A instrument for carrying out IF reactions
Clear Cell Culture Plate 24 well flat-bottom w/ lid BD 353047 plate used for growing monoclonal cells
CytoOne T25 filter cap TC flask USA Scientific CC7682-4825 container for growing mammalian cells
Dimethyl sulfoxide (DMSO) Fischer Scientific BP231-100 reagent used for freezing down engineered mammalian cells
Ethidium Bromide Thermo Fisher Scientific 15-585-011 reagent for gel electrophoresis
Falcon 96 Well Clear Flat Bottom TC-Treated Culture Microplate, with Lid Corning 353072 container for growing sorted monoclonal cells
FCS Express De Novo Software: N/A software for characterizing flow cytometry data
Fetal Bovine Serum, Regular, USDA 500 mL Corning 35-010-CV reagent for growing mammalian cells
Fisherbrand Petri Dishes with Clear Lid – Raised ridge; 100 x 15 mm Fisher Scientific FB0875712 equipment for growing bacteria
Gibco DMEM, High Glucose Thermo Fisher Scientific 11-965-092 reagent for growing mammalian cells
Hs00932330_m1 KRAS isoform a Taqman Gene Expression Assay Life Technologies 4331182 qPCR Probe
Hygromycin B (50 mg/mL), 20 mL Life Technologies 10687-010 reagent for selecting cells with proper gene circuit integration
iScript Reverse Transcription Supermix Bio-Rad Laboratories 1708890 reagent for converting RNA to cDNA
Laboratory Freezer -20 °C VWR 76210-392 equipment for storing experimental reagents
Laboratory Freezer -80 °C Panasonic MDF-U74VC equipment for storing experimental reagents
Laboratory Refrigerator +4 °C VWR 76359-220 equipment for storing experimental reagents
LB Broth (Lennox) , 1 kg Sigma-Aldrich L3022-250G reagent for growing bacteria
LIPOFECTAMINE 3000 Life Technologies L3000008 reagemt for transfecting gene circuits into mammalian cells
MATLAB 2019 MathWorks N/A software for analyzing experimental data
Methanol Acros Organics 413775000 reagent for immunofluorescence reaction
Microcentrifuge Tubes, Polypropylene 1.7 mL VWR 20170-333 plasticware container
Mr04097229_mr EGFP/YFP Taqman Gene Expression Assay Life Technologies 4331182 qPCR Probe
MultiTherm Shaker Benchmark Scientific H5000-HC equipment for bacterial transformation
NanoDrop Lite Spectrophotometer Thermo Fisher Scientific ND-NDL-US-CAN equipment for DNA/RNA concentration measurement
NEB Q5 High-Fidelity DNA polymerase 2x Master Mix NEB M0492S reagent for PCR of gene circuit fragments
NEB10-beta Competent E. coli (High Efficiency) New England Biolabs (NEB) C3019H bacterial cells for amplifying gene circuit of interest
NEBuilder HiFi DNA Assembly Master Mix New England Biolabs (NEB) E2621L reagent for combining gene circuit fragements
Nikon Eclipse Ti-E inverted microscope with a DS-Qi2 camera Nikon Instruments Inc. N/A instrument for quantifying gene expression
NIS-Elements Nikon Instruments Inc. N/A software for characterizing fluorescence microscopy data
oligonucleotides IDT N/A reagent used for PCR of gene circuit components
Panasonic MCO-170 AICUVHL-PA cellIQ Series CO2 Incubator with UV and H2O2 Control Panasonic MCO-170AICUVHL-PA instrument for growing mammalian cells
Paraformaldehyde, 16% Electron Microscopy Grade Electron Microscopy Sciences 15710-S reagent
PBS, Dulbecco's Phosphate-Buffered Saline (D-PBS) (1x) Invitrogen 14190144 reagent for mammalian cell culture,reagent for IF incubation buffer
Penicillin-Streptomycin (10,000 U/mL), 100x Fisher Scientific 15140-122 reagent for growing mammalian cells
primary ERK antibody Cell Signaling Technology 4370S primary ERK antibody for immunifluorescence
primary KRAS antibody Sigma-Aldrich WH0003845M1 primary KRAS antibody for immunifluorescence
QIAprep Spin Miniprep Kit (250) Qiagen 27106 reagent kit for purifying gene circuit plasmids
QIAquick Gel Extraction Kit (50) Qiagen 28704 reagent kit for purifying gene circuit fragments
QuantStudio 3 Real-Time PCR System Eppendorf A28137 equipment for qRT-PCR
Relative Quantification App Thermo Fisher Scientific N/A software for quantifying RNA/cDNA amplificaiton
RNeasy Plus Mini Kit Qiagen 74134 kit for extracting RNA of engineered mammalian cells
Secondary ERK antibody Cell Signaling Technology 8889S secondary ERK antibody for immunifluorescence
secondary KRAS antibody Invitrogen A11005 secondary KRAS antibody for immunifluorescence
Serological Pipets 5.0 mL Olympus Plastics 12-102 reagents used for setting up a variety of chemical reactions
SmartView Pro Imager System Major Science UVCI-1200 tool for imaging correct PCR bands
SnapGene Viewer (free) or SnapGene SnapGene N/A software DNA sequence design and analysis
Stage top incubator Tokai Hit INU-TIZ tool for carrying PCR, transformation, or gel extraction reactions
TaqMan Fast Advanced Master Mix Thermo Fisher Scientific 4444557 reagent for PCR of gene circuit fragments
TaqMan Human GAPD (GAPDH) Endogenous Control (VIC/MGB probe), primer limited, 2500 rxn Life Technologies 4326317E qPCR Probe
Thermocycler Bio-Rad 1851148 tool for carrying PCR, transformation, or gel extraction reactions
VisiPlate-24 Black, Black 24-well Microplate with Clear Bottom, Sterile and Tissue Culture Treated PerkinElmer 1450-605 plate used for light-induction experiments
VWR Disposable Pasteur Pipets, Glass, Borosilicate Glass Pipet, Short Tip, Capacity=2 mL, Overall Length=14.6 cm VWR 14673-010 reagent for mammalian cell culture
VWR Mini Horizontal Electrophoresis Systems, Mini10 Gel System VWR 89032-290 equipment for DNA gel electrophoresis
Flp-In 293 Thermo Fisher Scientific R75007 Engineered cell line with FRT site
DOWNLOAD MATERIALS LIST

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OptogeneticsGene CircuitsMammalian CellsSpatial-temporal ControlCell EngineeringGene ExpressionCloningTransfectionSingle-cell SortingMonoclonal CellsLPA Circuit3D Printed PartsIris SoftwareLight Stimulation
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Guinn, M. T., Coraci, D., Guinn, L., Balázsi, G. Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells. J. Vis. Exp. (173), e62109, doi:10.3791/62109 (2021).
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