Mammalian Cell Engineering: Methods and Protocols

Preface
Contents
Contributors
Part I: Engineering Mammalian Cells to Sense Biologically Relevant Inputs
	Chapter 1: Generation of CAR-T Cells by Lentiviral Transduction
		1 Introduction
		2 Materials
			2.1 Vectors (See Note 1)
			2.2 Packaging Cell Transfection
			2.3 Virus Concentration
			2.4 T Cell Isolation
			2.5 T Cell Culture and Transduction
			2.6 Validation Assay
		3 Methods
			3.1 Packaging Cell Transfection
			3.2 Virus Concentration (See Note 9)
			3.3 T Cell Isolation
			3.4 T Cell Transduction
			3.5 Validation Assay
		4 Notes
		References
	Chapter 2: Synthetic Receptors for Sensing Soluble Molecules with Mammalian Cells
		1 Introduction
		2 Materials
		3 Methods
			3.1 Transient Transfections of HEK293T Cells
			3.2 Generating Stable Cell Lines
		4 Notes
		References
	Chapter 3: Engineering Mammalian Cells to Control Glucose Homeostasis
		1 Introduction
			1.1 Closed-Loop Control Devices-Electronic vs. Cell-Based Approaches
			1.2 Clinically Relevant Treatment Strategies-Closed- Loop vs. Open-Loop Systems
		2 Materials
			2.1 Plasmids for Synthetic Gene Circuits
			2.2 Host Cell Culture and Transfection
			2.3 Encapsulation Material
			2.4 Animal Experiments
			2.5 Analytical Assays
		3 Methods
			3.1 Synthetic Gene Circuits
				3.1.1 Defining the Engineering Goals
				3.1.2 Validation and Optimization of the Circuit
			3.2 Manufacture of Designer Cells
			3.3 Efficacy Testing In Vivo for Control of Glucose Homeostasis
				3.3.1 Preparation of Alginate-Poly-(l-Lysine)-Alginate Beads
				3.3.2 Animal Experiment
		4 Notes
		References
	Chapter 4: Using Engineered Mammalian Cells for an Epitope-Directed Antibody Affinity Maturation System
		1 Introduction
		2 Materials
			2.1 Vector Construction
			2.2 Establishment of the Engineered Cells
			2.3 Library Preparation and Screening
		3 Methods
			3.1 Vector Construction
			3.2 Transduction of Vectors Coding the Antigen/Fas Chimera and Dimerizing Antibody to Ba/F3 Cells (See Note 7)
			3.3 Cell Cloning (See Note 8)
			3.4 Evaluation of Cloned Cells Expressing the Antigen/Fas Chimera and Dimerizing Antibody (See Note 7)
			3.5 Preparation of a Plasmid Coding an scFv Library
			3.6 Library Screening (See Note 7)
		4 Notes
		References
	Chapter 5: Purification of Specific Cell Populations Differentiated from Stem Cells Using MicroRNA-Responsive Synthetic Messen...
		1 Introduction
		2 Materials
			2.1 Template DNA Preparation by PCR
			2.2 mRNA Preparation by In Vitro Transcription
			2.3 Cell Culture
			2.4 mRNA Transfection
			2.5 Flow Cytometry
		3 Methods
			3.1 Choice of Appropriate Fluorescent Proteins
			3.2 Design of Primers for ORF PCR
			3.3 Template DNA Preparation by PCR
			3.4 mRNA Synthesis by In Vitro Transcription
			3.5 mRNA Transfection and Flow Cytometry
		4 Notes
		References
Part II: Engineering Mammalian Cells to Sense Artificial Inputs
	Chapter 6: Green Light-Controlled Gene Switch for Mammalian and Plant Cells
		1 Introduction
		2 Materials
			2.1 Cloning of Multiple CarO Repeats
			2.2 Mammalian Cell Culture, Transfection, and Light Treatment
			2.3 Quantification of mRNA Expression in Mammalian Cells
			2.4 Quantification of SEAP Expression in Mammalian Cells
			2.5 Reporter Gene Expression in Protoplasts
				2.5.1 Protoplast Preparation
				2.5.2 Protoplast Transformation, Light Treatment, and Luciferase Assay
		3 Methods
			3.1 Cloning of Multiple CarO Repeats
			3.2 Mammalian Cell Culture, Transfection, Light Incubation, and Harvest
			3.3 Quantification of SEAP Expression in Mammalian Cells
			3.4 Quantification of mRNA Expression in Mammalian Cells
			3.5 Protoplast Preparation
			3.6 Protoplast Transformation, Light Treatment, and Luciferase Assay
		4 Notes
		References
	Chapter 7: Sonogenetic Modulation of Cellular Activities in Mammalian Cells
		1 Introduction
		2 Materials
			2.1 US Apparatus
			2.2 Live-Cell Imaging System
			2.3 Cell Culture and Transfection
			2.4 Construction of Plasmid DNAs
			2.5 Preparation of MBs
			2.6 Animal Experiments
			2.7 Immunohistochemical Staining
		3 Methods
			3.1 Introducing Two Mutants into Mouse Prestin DNA
			3.2 DNA Transfection
			3.3 Preparation of DNA-Loaded MBs
			3.4 Adeno-Associated Virus Production
			3.5 Acoustic Peak Negative Pressure and US Focal Zone Measurement
			3.6 In Vitro US Stimulation
			3.7 Live-Cell Imaging and Data Analysis
			3.8 In Vivo Gene Delivery
				3.8.1 Adeno-Associated Virus Infection
				3.8.2 Sonotransfection
			3.9 In Vivo US Stimulation
			3.10 Immunohistochemical Staining
		4 Notes
		References
	Chapter 8: Constructing Smartphone-Controlled Optogenetic Switches in Mammalian Cells
		1 Introduction
		2 Materials
			2.1 Construction of the Smartphone Controlled FRL-Responsive Module
			2.2 Construction of the Plasmids
				2.2.1 Instruments
				2.2.2 Reagents
			2.3 Cell Culture and Transfection
				2.3.1 Instruments
				2.3.2 Reagents
			2.4 FRL Illumination
			2.5 SEAP Reporter Assay
				2.5.1 Instruments
				2.5.2 Reagents
		3 Methods
			3.1 Construction the Smartphone Controlled FRL-Responsive Module (SmartController 1.0)
				3.1.1 Development of the Smartphone ECNU-TeleMed app
				3.1.2 Assembly of the far-red LED Array Module
				3.1.3 Assembly of the SmartController 1.0
			3.2 Construction of the BphS-BldD Based FRL-Triggered Optogenetic Switch
			3.3 Cell Culture and Transfection
			3.4 FRL Illumination
			3.5 SEAP Reporter Assay
		4 Notes
		References
	Chapter 9: Constructing a Smartphone-Controlled Semiautomatic Theranostic System for Glucose Homeostasis in Diabetic Mice
		1 Introduction
		2 Materials
			2.1 Plasmids (Table 1)
			2.2 Buffers and Stock Solutions
			2.3 Lab Equipment
			2.4 Cells and Animals
		3 Methods
			3.1 Construction of the SmartController 3.0
				3.1.1 Custom-Designed Glucometer
				3.1.2 Blood Glucose Microprocessor
				3.1.3 SmartControl-Box 3.0
				3.1.4 Custom-Designed Electromagnetic Emission Circuit (EEC)
				3.1.5 Coiled-LEDs
			3.2 Construction of the Stable Cell Line HEKFRL-SEAP-P2A-mINS and HEKFRL-shGLP-1-P2A-SEAP
			3.3 Construction of the Type 1 Diabetic Mouse Model
			3.4 HydrogeLED Implant
				3.4.1 Preparation of the hydrogeLED Implant
				3.4.2 HydrogeLED Implantation
			3.5 Semiautomatic Control of Blood Glucose Homeostasis in Diabetic  Mice
			3.6 The Therapeutic Efficacy of the Smartphone-Regulated Semiautomatic Theranostic System in Diabetic  Mice
				3.6.1 Insulin ELISA
				3.6.2 Glucagon-Like Peptide-1 (GLP-1) ELISA
				3.6.3 Intraperitoneal Glucose Tolerance Test (IGTT) in  Mice
				3.6.4 Intraperitoneal Insulin Tolerance Test (IGTT) in  Mice
		4 Notes
		References
	Chapter 10: Construction of Caffeine-Inducible Gene Switches in Mammalian Cells
		1 Introduction
			1.1 Inducible Gene Expression Systems in Mammalian Synthetic Biology
			1.2 Using Caffeine in Mammalian Synthetic Biology
		2 Materials
			2.1 Cell Culture
			2.2 SEAP Reporter Gene Expression Assay
		3 Methods
			3.1 Design Principles for Constructing a Caffeine-Inducible Gene Expression System
			3.2 Construct Plasmids for a Caffeine-Inducible Gene Expression System
			3.3 Cell Cultivation and Transfection
			3.4 Testing the Caffeine-Inducible Gene Expression System
		4 Notes
		References
Part III: Precise Genome Engineering Techniques Using CRISPR-Cas Systems
	Chapter 11: Multiplexed Genome Engineering with Cas12a
		1 Introduction
		2 Materials
			2.1 Plasmids
			2.2 Molecular Reagents
			2.3 Kits
			2.4 Enzymes
			2.5 Human Cell Culture
			2.6 Special Equipment
			2.7 Sequencing Primers
		3 Methods
			3.1 Construction of Customized SiT-Cas12a Effector Plasmids
			3.2 Generation and Incorporation of crRNA Arrays into SiT-Cas12a Plasmids
				3.2.1 Assembly and Incorporation of Small crRNA Arrays into SiT-Cas12a Plasmids
				3.2.2 Assembly and Incorporation of Medium crRNA Arrays into SiT-Cas12a Plasmids
				3.2.3 Assembly and Incorporation of Large crRNA Arrays into SiT-Cas12a Plasmids
			3.3 Maintenance and Transfection of HEK 293T Cells
			3.4 Analysis of SiT-Cas12a-mediated Genome Editing Efficiency
			3.5 Validation of SiT-Cas12a-Mediated Transcriptional Regulatory Efficiency
		4 Notes
		References
	Chapter 12: Highly Multiplexed Analysis of CRISPR Genome Editing Outcomes in Mammalian Cells
		1 Introduction
		2 Materials
			2.1 General Considerations
			2.2 Screening of gRNA Target Sites and Target Amplification Primers
			2.3 Preparation of gRNA Expression Plasmid Library
			2.4 Culturing of Cells
			2.5 Massively Parallel Transfection of Genome-Editing Reagents
			2.6 Template Genomic DNA Preparation
			2.7 Generation of a Multiplexed Amplicon Sequencing Library and Sequencing
			2.8 List of Equipment
		3 Methods
			3.1 Screening of gRNA Target Sites and Amplification Primers
				3.1.1 Primer Preparation
				3.1.2 Template Genomic DNA Preparation
				3.1.3 Primer Screening by  qPCR
			3.2 Preparation of gRNA Expression Plasmid Library
				3.2.1 Preparation of gRNA Spacer Inserts
				3.2.2 Ligation Assembly
				3.2.3 Bacterial Transformation
				3.2.4 Plasmid Purification and Sanger Sequencing
			3.3 Culturing of Cells
				3.3.1 Collagen Coating of Cell Culture Plates
				3.3.2 Seeding Cells into 96-Well Collagen-Coated Cell Culture Plates
			3.4 Massively Parallel Transfection of Genome-Editing Reagents
				3.4.1 gRNA Reagent Preparation
				3.4.2 Transfection
			3.5 Template Genomic DNA Preparation
			3.6 Generation of a Multiplexed Amplicon Sequencing Library and Sequencing
				3.6.1 PCR Amplification of the Target Regions
				3.6.2 Purification of the First PCR Products
				3.6.3 Pooled Indexing PCR for Multiplexed Sequencing
				3.6.4 Multiplexed Amplicon Sequencing
		4 Notes
		References
	Chapter 13: Optical Control of Genome Editing by Photoactivatable Cas9
		1 Introduction
		2 Materials
			2.1 Media and Buffers
			2.2 Molecular Biology
			2.3 Plasmids
			2.4 Cell and Transfection Reagents
			2.5 Instruments
		3 Methods
			3.1 Generation of Single-Guide RNA (sgRNA) Expression Vector (Fig. 2)
			3.2 Luciferase Plasmid HDR Assay (Fig. 3)
			3.3 Optogenetic Genome Editing Experiments
			3.4 T7 Endonuclease I (T7EI) Assay for Quantifying Indel Mutation of Endogenous Genes (Fig. 4)
			3.5 RFLP Assay for Detecting HDR-Mediated Modification in Endogenous Human  Gene
		4 Notes
		References
Part IV: Engineering Mammalian Cells in Combination with Chemical Compounds/Systems
	Chapter 14: Chemogenetic Control of Protein Localization and Mammalian Cell Signaling by SLIPT
		1 Introduction
		2 Materials
			2.1 Solid-Phase Synthesis of mDcTMP
			2.2 Plasmids
			2.3 Cell Culture and Transfection
			2.4 Live-Cell Fluorescence Imaging
			2.5 SLIPT Experiments
		3 Methods
			3.1 Synthesis of mDcTMP
			3.2 Preparation of mDcTMP Stock Solution
			3.3 Preparation of TMP Stock Solution
			3.4 Reversible SLIPT of EGFP-DHFRiK6
				3.4.1 Cell Seeding and Transfection
				3.4.2 Reversible SLIPT and Fluorescence Imaging
			3.5 SLIPT-Mediated cRaf Translocation and ERK Activation
				3.5.1 Cell Seeding and Transfection
				3.5.2 SLIPT-Mediated Activation of the cRaf/ERK Pathway
		4 Notes
		References
	Chapter 15: Engineering Hydrogel Production in Mammalian Cells to Synthetically Mimic RNA Granules
		1 Introduction
			1.1 A Synthetic Biological Approach to Intracellular Assembly of Multiple Biomolecules
			1.2 Growing Palette of Synthetic Biology Tools in the Field
			1.3 Advantages and Disadvantages of iPOLYMER
			1.4 Requirement for Appropriate Control Experiments
		2 Materials
			2.1 Materials for Cell Culture
			2.2 Materials for Transfection
			2.3 Materials for Live-Cell Fluorescence Imaging
			2.4 Materials for Immunostaining Against Stress Granule Markers and a Nonmarker
		3 Methods
			3.1 Induce iPOLYMER Condensate Formation in Living Cells by Chemical Stimulus
				3.1.1 Preparing Rapamycin Stock
				3.1.2 Validating the Stock by CID-Dependent Protein Translocation
				3.1.3 Live-Cell Imaging of iPOLYMER Condensate Formation in Living Cells
				3.1.4 Immunostaining of Fixed Cells with iPOLYMER Condensates
			3.2 Live-Cell Imaging of iPOLYMER-LI Condensate Formation
			3.3 Immunostaining of Fixed Cells with Light-Induced iPOLYMER-LI Condensates
		4 Notes
		References
	Chapter 16: AgDD System: A Chemical Controllable Protein Aggregates in Cells
		1 Introduction
		2 Materials
			2.1 Establishing AgDD Cell Lines
			2.2 Analyzing AgDD Cell Lines
			2.3 Recombinant Protein Purification: BL21 Cells Expressing His6-FKBP(F36V) Protein
		3 Methods
			3.1 Cell Culture
			3.2 Preparing Purified Recombinant Protein for the Experiments
			3.3 Establishing AgDD Cell Lines
			3.4 Inducing Protein Aggregates
			3.5 Analyzing the Protein Aggregates in FACS
			3.6 Analyzing the Protein Aggregates Under Fluorescence Microscopy
		4 Notes
		References
	Chapter 17: Intracellular Unnatural Catalysis Enabled by an Artificial Metalloenzyme
		1 Introduction
		2 Materials
			2.1 Reagents Used for Preparation of an Artificial Deallylase and for Intracellular Catalysis
			2.2 Plasmids and Transfection Reagents
			2.3 Reagents for Cell Culture
			2.4 Reagents for the Enzymatic Activity Assays
			2.5 Instruments and Consumables
		3 Method
			3.1 Preparation of Sav Stock Solution with 800 μM  FBBS
			3.2 Preparation of Artificial Deallylase 1xSav
			3.3 Preparation of Cell-Penetrating Artificial Deallylase 1x2ySav
			3.4 Cell Culture and Transfection (Fig. 4)
			3.5 Intracellular Catalysis of Artificial Deallylase 1x2ySav (Fig. 4)
			3.6 Sec-nluc Activity Assay (Fig. 4)
			3.7 SEAP Activity Assay (Fig. 4)
		4 Notes
		References
	Chapter 18: Feeder-Free Human Induced Pluripotent Stem Cell Culture Using a DNA Aptamer-Based Mimic of Basic Fibroblast Growth...
		1 Introduction
		2 Materials
			2.1 Cell Culture Medium
		3 Methods
			3.1 Preparation of the Aptamer Stock Solution
			3.2 Maintenance of Human iPSCs in Culture Medium Containing the bFGF-Mimicking Aptamer
		4 Notes
		References
Part V: New Techniques to Engineer Specific Mammalian Cells in a Targeted Manner
	Chapter 19: Protocol for De Novo Gene Targeting Via In Utero Electroporation
		1 Introduction
		2 Materials
		3 Methods
			3.1 Plasmid Construction
			3.2 Donor Vector Construction
			3.3 Guide RNA Vector Construction
			3.4 In Utero Electroporation
		4 Notes
		References
	Chapter 20: Magnetically Single-Cell Virus Stamping
		1 Introduction
		2 Materials
			2.1 Biological Materials
			2.2 Reagents
			2.3 Equipment
		3 Method
			3.1 Preparing Virus-Bound Magnetic Nanoparticles
			3.2 Calibrating the Cell Culture Chamber
			3.3 Calibrating the Magnet for Nanoparticle Pullout and Assessing Gene Expression
		4 Notes
		References
Index