A Laboratory Guide to the Tight Junction

A Laboratory Guide to the Tight Junction
Copyright
Dedication
Contents
Author biography
Preface
Acknowledgments
1 Introduction
1.1 Cell junction
	1.1.1 Junction category
	1.1.2 Tight junction
	1.1.3 Adherens junction
	1.1.4 Desmosome
	1.1.5 Gap junction
	References
1.2 Cell adhesion
	1.2.1 Cadherin interaction
	1.2.2 Claudin interaction
	1.2.3 Connexin interaction
	1.2.4 Cadherin compatibility and tissue morphogenesis
	1.2.5 Claudin compatibility and tissue barrier
	1.2.6 Connexin compatibility and electric synapse
	References
1.3 Paracellular channel
	1.3.1 Ion channel in tight junction
	1.3.2 Electric conductance of paracellular channel
	1.3.3 Ion selectivity of paracellular channel
	1.3.4 Size selectivity of paracellular channel
	1.3.5 Paracellular water channel
	References
1.4 Perijunctional cytoskeleton
	1.4.1 Actin polymerization
	1.4.2 Actin reorganization
	1.4.3 Contractile apparatus
	1.4.4 Mechanosensitive signal transduction
	1.4.5 Contractility and paracellular permeability
	References
1.5 Junction signaling
	1.5.1 Catenin signaling
	1.5.2 Small GTPases
	1.5.3 Receptor tyrosine kinases
	1.5.4 Canonical Wnt signaling
	1.5.5 Hippo signaling
	References
2 Biochemical approaches for tight junction
2.1 Biochemistry of tight junction
	2.1.1 Biochemical organization of tight junction
	2.1.2 Tight junction enriched protein fraction
	2.1.3 Tight junction integral protein fraction
	2.1.4 Molecular structure of claudin protein
	2.1.5 Models of claudin interaction
	2.1.6 Claudin interaction with tight junction plaque proteins
	2.1.7 Tight junction anchorage onto cytoskeleton
	References
2.2 Tight junction isolation by subcellular fractionation
	2.2.1 Background knowledge
		2.2.1.1 Subcellular fractionation
		2.2.1.2 Detergent and denaturant extraction
	2.2.2 Materials and reagents
		2.2.2.1 Animals
		2.2.2.2 Homogenizer
		2.2.2.3 Centrifuge
		2.2.2.4 Buffers
	2.2.3 Experimental procedure
	2.2.4 Data analysis
	2.2.5 Troubleshooting
		2.2.5.1 Contamination of intracellular membrane
		2.2.5.2 Contamination of lipid-raft microdomains
		2.2.5.3 Contamination of adherens junction
		2.2.5.4 Extrapolation to other organ systems
	2.2.6 Concluding remarks
	References
2.3 Immunoprecipitation of cis and trans claudin interactions
	2.3.1 Background knowledge
		2.3.1.1 Coimmunoprecipitation
		2.3.1.2 Preservation of protein interaction
		2.3.1.3 Cis versus trans interaction
	2.3.2 Materials and reagents
		2.3.2.1 Cell model for ectopic gene expression
		2.3.2.2 Plasmids
		2.3.2.3 Cell culture medium
		2.3.2.4 Buffers
	2.3.3 Experimental procedure
		2.3.3.1 Immunoprecipitation of cis claudin interactions
		2.3.3.2 Immunoprecipitation of trans claudin interactions
	2.3.4 Data analysis
	2.3.5 Troubleshooting
		2.3.5.1 Lysis condition
		2.3.5.2 Choice of antibody
		2.3.5.3 Nonspecific protein interactions
	2.3.6 Concluding remarks
	References
2.4 Isolation of claudin oligomer by chemical cross-linking
	2.4.1 Background knowledge
		2.4.1.1 Chemical cross-linkers
		2.4.1.2 Ectopic claudin expression model
	2.4.2 Materials and reagents
		2.4.2.1 Cell model for ectopic gene expression
		2.4.2.2 Plasmids
		2.4.2.3 Cell culture medium
		2.4.2.4 Buffers
		2.4.2.5 Chemical cross-linkers
		2.4.2.6 Equipment
	2.4.3 Experimental procedure
		2.4.3.1 HEK293 cell transfection
		2.4.3.2 Solubilization of claudin protein
		2.4.3.3 Sucrose gradient centrifugation
		2.4.3.4 Chemical cross-linking
	2.4.4 Data analysis
	2.4.5 Troubleshooting
		2.4.5.1 Specificity of cross-linking
		2.4.5.2 Molecular ruler and nearest neighbor approaches
		2.4.5.3 In vitro versus in vivo cross-linking
		2.4.5.4 cis versus trans oligomer
	2.4.6 Concluding remarks
	References
2.5 Yeast two-hybrid assay of claudin interaction
	2.5.1 Background knowledge
		2.5.1.1 Classic yeast two-hybrid assay
		2.5.1.2 Membrane yeast two-hybrid assay
	2.5.2 Materials and reagents
		2.5.2.1 Saccharomyces cerevisiae yeast strain
		2.5.2.2 Plasmids
		2.5.2.3 Yeast growth media
	2.5.3 Experimental procedure
		2.5.3.1 Transformation of bait and prey constructs into NMY51 yeast strain
		2.5.3.2 Verification of bait and prey protein expression by Western blot
		2.5.3.3 Dual transformation and reporter gene expression assay
	2.5.4 Data analysis
		2.5.4.1 Protein topology
		2.5.4.2 Quantitative β-galactosidase assay
		2.5.4.3 Ade2 phenotype
		2.5.4.4 His3 reporter stringency test
	2.5.5 Troubleshooting
		2.5.5.1 False-positive interaction
		2.5.5.2 Protein stability in Saccharomyces cerevisiae
	2.5.6 Closing remarks
	References
2.6 Recombinant claudin protein production in Pichia pastoris
	2.6.1 Background knowledge
		2.6.1.1 Recombinant protein expression system
			2.6.1.1.1 Prokaryotes
			2.6.1.1.2 Eukaryotes
		2.6.1.2 Chromatography
	2.6.2 Materials and reagents
		2.6.2.1 Pichia pastoris yeast strain
		2.6.2.2 Plasmids
		2.6.2.3 Yeast growth media
		2.6.2.4 Pichia pastoris EasyComp transformation kit
		2.6.2.5 Chromatography
	2.6.3 Experimental procedure
		2.6.3.1 Pichia pastoris transformation
			2.6.3.1.1 Prepare competent Pichia pastoris cells
			2.6.3.1.2 Transformation
		2.6.3.2 Pichia pastoris induction
		2.6.3.3 HisGFP-claudin purification from Pichia pastoris
			2.6.3.3.1 Lysis and extraction
			2.6.3.3.2 Affinity chromatography
			2.6.3.3.3 Size-exclusion chromatography
	2.6.4 Data analysis
	2.6.5 Troubleshooting
		2.6.5.1 Protein solubilization
		2.6.5.2 Optimization of imidazole concentration for IMAC Ni-charged affinity chromatography
		2.6.5.3 Reducing nonspecific protein interaction in IMAC Ni-charged affinity chromatography
		2.6.5.4 pH and EDTA levels in IMAC Ni-charged affinity chromatography
		2.6.5.5 Molecular weight and shape in size-exclusion chromatography
		2.6.5.6 Resolution in size-exclusion chromatography
	2.6.6 Closing remarks
	References
3 Biophysical approaches for tight junction
3.1 Electrophysiology of epithelial transport
	3.1.1 Electric potential, resistance, and capacitance of cell membrane
		3.1.1.1 Membrane potential
		3.1.1.2 Membrane resistance
		3.1.1.3 Membrane capacitance
	3.1.2 Basic principles of cell membrane electrophysiology
		3.1.2.1 Equivalent electrical circuit of cell membrane
		3.1.2.2 Electric current
		3.1.2.3 Ohm’s law
		3.1.2.4 Voltage divider
		3.1.2.5 Current through capacitor
		3.1.2.6 Electrode, liquid junction potential, and salt bridge
	3.1.3 Electrophysiology of an epithelium
		3.1.3.1 Equivalent electrical circuit of an epithelium
		3.1.3.2 Transepithelial resistance
		3.1.3.3 Transepithelial potential
	3.1.4 Noise prevention and signal conditioning
		3.1.4.1 External noise
		3.1.4.2 Intrinsic noise
		3.1.4.3 Filtering
	3.1.5 Data acquisition and digitization
	References
3.2 Epithelial cell cultures in Ussing chamber
	3.2.1 Background knowledge
		3.2.1.1 Theoretic considerations
			3.2.1.1.1 Recording of transcellular transport
			3.2.1.1.2 Recording of paracellular transport
		3.2.1.2 Practical applications
			3.2.1.2.1 Classic Ussing chamber
			3.2.1.2.2 Self-contained Ussing chamber
			3.2.1.2.3 Transwell permeable supports
	3.2.2 Materials and instrumentation
		3.2.2.1 Electrophysiological rig
		3.2.2.2 Ussing chamber assembly
		3.2.2.3 Superfusate
		3.2.2.4 Cell culture
	3.2.3 Experimental procedure
		3.2.3.1 Setting up Ussing chamber
		3.2.3.2 Measuring the short-circuit current
		3.2.3.3 Measuring the paracellualr conductance
		3.2.3.4 Measuring the paracellular ion selectivity
	3.2.4 Data analysis
		3.2.4.1 Baseline and peak amplitude
		3.2.4.2 I–V curve
		3.2.4.3 MATLAB functions
	3.2.5 Troubleshooting
		3.2.5.1 Quality of cell monolayer
		3.2.5.2 Quality of electrodes
		3.2.5.3 Electric pulse
	3.2.6 Closing remarks
	References
3.3 Epithelial tissues in Ussing chamber
	3.3.1 Background knowledge
		3.3.1.1 Intestinal epithelium
		3.3.1.2 Renal epithelium
	3.3.2 Materials and instrumentation
		3.3.2.1 Electrophysiological rig
		3.3.2.2 Ussing chamber assembly
		3.3.2.3 Superfusate
		3.2.2.4 Mouse colon preparation
	3.3.3 Experimental procedure
		3.3.3.1 Setting up Ussing chamber
		3.3.3.2 Measuring the short-circuit current
		3.3.3.3 Measuring the paracellualr conductance
	3.3.4 Data analysis
		3.3.4.1 MATLAB functions
		3.3.4.2 Electrical contribution from multiple tissue layers
	3.3.5 Troubleshooting
		3.3.5.1 Edge damage
		3.3.5.2 Tissue viability and variability
		3.3.5.3 Electric pulse
	3.3.6 Closing remarks
	References
3.4 Epithelial ohmmeter
	3.4.1 Background knowledge
		3.4.1.1 “Chopstick” electrode system
		3.4.1.2 Current clamp and Ohm’s law
	3.4.2 Materials and instrumentation
	3.4.3 Experimental procedure
	3.4.4 Data analysis
	3.4.5 Troubleshooting
		3.4.5.1 Nonuniform electric field
		3.4.5.2 Cell capacitance and underestimation of Rte
	3.4.6 Closing remarks
	References
3.5 Impedance measurement in Ussing chamber
	3.5.1 Background knowledge
		3.5.1.1 Concept of impedance measurement
		3.5.1.2 Sinusoidal current waveform
		3.5.1.3 Impedance of resistor and capacitor
		3.5.1.4 Nyquist plot
	3.5.2 Materials and instrumentation
		3.5.2.1 Electrophysiological rig
		3.5.2.2 Buffer
		3.5.2.3 Cell culture
	3.5.3 Experimental procedure
	3.5.4 Data analysis
	3.5.5 Troubleshooting
		3.5.5.1 Sample-electrode distance
		3.5.5.2 Phase shift
		3.5.5.3 Paracellular versus transcellular pathway
	3.5.6 Closing remarks
	References
3.6 Flux assay in Ussing chamber
	3.6.1 Background knowledge
		3.6.1.1 Fick’s law
		3.6.1.2 Radioisotope
	3.6.2 Materials and instrumentation
		3.6.2.1 Buffer
		3.6.2.2 Cell culture
		3.6.2.3 Liquid scintillation counter
		3.6.2.4 Radioisotope
	3.6.3 Experimental procedure
	3.6.4 Data analysis
	3.6.5 Troubleshooting
		3.6.5.1 Radiation safety
		3.6.5.2 Differentiation of paracellular from transcellular pathway
	3.6.6 Closing remarks
	References
3.7 Measurement of water permeability in Ussing chamber
	3.7.1 Background knowledge
		3.7.1.1 Transepithelial water permeability
		3.7.1.2 A simplified model
	3.7.2 Materials and instrumentation
		3.7.2.1 Ussing chamber perfusion rig
		3.7.2.2 Superfusate
		3.7.2.3 Cell culture
	3.7.3 Experimental procedure
		3.7.3.1 Cancelation of hydrostatic pressure
		3.7.3.2 Measuring transepithelial water permeability
	3.7.4 Data analysis
	3.7.5 Troubleshooting
		3.7.5.1 Differentiating paracellular from transcellular water pathway
		3.7.5.2 Effect of transepithelial voltage
		3.7.5.3 Proton permeability
		3.7.5.4 Limitation in direct measurement of volume
	3.7.6 Closing remarks
	References
4 Histological approaches for tight junction
4.1 Fixation and fixatives
	4.1.1 Classification of fixatives
	4.1.2 Mechanism of fixation
		4.1.2.1 Protein cross-linking and denaturation
			4.1.2.1.1 Cross-link formation
			4.1.2.1.2 Denaturation
		4.1.2.2 Lipid oxidization and cross-linking
		4.1.2.3 Reaction of fixatives with nucleic acids
	4.1.3 Concentration of fixatives
	4.1.4 Osmolality of fixative solution
	4.1.5 Penetration of fixatives
	4.1.6 Temperature of fixation
	4.1.7 Duration of fixation
	4.1.8 Fixation artifacts
	References
4.2 Fixation
	4.2.1 Introduction
	4.2.2 Materials and reagents
	4.2.3 Experimental procedure
		4.2.3.1 Perfusion fixation through the heart
		4.2.3.2 Perfusion fixation through the abdominal aorta
		4.2.3.3 Immersion fixation
	4.2.4 Data analysis
	4.2.5 Troubleshooting
	4.2.6 Concluding remarks
	References
4.3 Tight junction atlas
	4.3.1 Introduction
	4.3.2 Survey of tight junction in organ systems
		4.3.2.1 Cardiovascular system
		4.3.2.2 Skin
		4.3.2.3 Lung
		4.3.2.4 Gastrointestinal tract
		4.3.2.5 Liver
		4.3.2.6 Kidney
		4.3.2.7 Nerve
	4.3.3 Concluding remarks
	References
5 Light microscopy for tight junction
5.1 Theory of light microscopy
	5.1.1 Lateral resolution in light microscopy
	5.1.2 Axial resolution in light microscopy
	5.1.3 Depth of field in light microscopy
	5.1.4 Fluorescence microscopy
	5.1.5 Fluorescent labels
	5.1.6 Autofluorescence
	5.1.7 Photobleaching
	References
5.2 Wide-field fluorescence microscopy for cells on cover glass
	5.2.1 Background knowledge
		5.2.1.1 Immunofluorescence labeling
		5.2.1.2 Fixation and permeabilization
	5.2.2 Materials and reagents
		5.2.2.1 Equipment
		5.2.2.2 Cell model
		5.2.2.3 Cell culture medium
		5.2.2.4 Buffers
	5.2.3 Experimental procedure
	5.2.4 Data analysis
	5.2.5 Troubleshooting
		5.2.5.1 Fixation artifact
		5.2.5.2 Antibody specificity
		5.2.5.3 Antibody avidity
		5.2.5.4 Double or triple immunofluorescence labeling
		5.2.5.5 Limit of resolution
	5.2.6 Concluding remarks
	References
5.3 Wide-field fluorescence microscopy for thin tissue section
	5.3.1 Background knowledge
		5.3.1.1 Cryostat section versus paraffin section
		5.3.1.2 Cryostat sectioning
			5.3.1.2.1 Freezing of fresh unfixed tissue
			5.3.1.2.2 Microtome temperature
			5.3.1.2.3 Sectioning technique
			5.3.1.2.4 Postsectioning fixation
	5.3.2 Materials and reagents
		5.3.2.1 Equipment
		5.3.2.2 Tissue-tek
		5.3.2.3 Animals
		5.3.2.4 Buffers
	5.3.3 Experimental procedure
		5.3.3.1 Freezing tissues
		5.3.3.2 Cutting cryostat sections
		5.3.3.3 Immunolabeling cryostat sections
	5.3.4 Data analysis
	5.3.5 Troubleshooting
		5.3.5.1 Fixation and tight junction pattern
		5.3.5.2 Nonspecific antibody binding
	5.3.6 Concluding remarks
	References
5.4 Confocal microscopy for cells on Transwell
	5.4.1 Background knowledge
		5.4.1.1 Confocal microscopy
		5.4.1.2 Apicobasal polarity
		5.4.1.3 Fixation of tight junction
	5.4.2 Materials and reagents
		5.4.2.1 Equipment
		5.4.2.2 Cell model
		5.4.2.3 Cell culture medium
		5.4.2.4 Buffers
	5.4.3 Experimental procedure
	5.4.4 Data analysis
	5.4.5 Troubleshooting
		5.4.5.1 Signal sensitivity
		5.4.5.2 Axial resolution
		5.4.5.3 Live-cell imaging
	5.4.6 Concluding remarks
	References
5.5 Confocal microscopy for thick tissue sections
	5.5.1 Background knowledge
	5.5.2 Materials and reagents
		5.5.2.1 Equipment
		5.5.2.2 Tissue-tek
		5.5.2.3 Animals
		5.5.2.4 Buffers
	5.5.3 Experimental procedure
	5.5.4 Data analysis
	5.5.5 Troubleshooting
	5.5.6 Concluding remarks
	References
6 Electron microscopy for tight junction
6.1 Theory of electron microscopy
	6.1.1 Wave-particle duality of electron
	6.1.2 Electromagnetic lens
	6.1.3 Specimen preparation
	6.1.4 Ultramicrotomy
	6.1.5 Positive staining
	6.1.6 Negative staining
	6.1.7 Low temperature methods
	6.1.8 Immunolabeling techniques
	References
6.2 Transmission electron microscopy for cell culture
	6.2.1 Background knowledge
		6.2.1.1 Electron microscopy for tight junction
		6.2.1.2 Fixation for electron microscopy
	6.2.2 Materials and reagents
		6.2.2.1 Equipment
		6.2.2.2 Cell model
		6.2.2.3 Cell culture medium
		6.2.2.4 Buffers
	6.2.3 Experimental procedure
		6.2.3.1 Cell culture and fixation
		6.2.3.2 Embedding
		6.2.3.3 Sectioning and poststaining
	6.2.4 Data analysis
	6.2.5 Troubleshooting
		6.2.5.1 Fixation of membrane structure
		6.2.5.2 Poststaining of tight junction
	6.2.6 Concluding remarks
	References
6.3 Transmission electron microscopy for tissue section
	6.3.1 Background knowledge
		6.3.1.1 Tissue perfusion
		6.3.1.2 Tissue fixation
	6.3.2 Materials and reagents
		6.3.2.1 Equipment
		6.3.2.2 Buffers
	6.3.3 Experimental procedure
		6.3.3.1 Perfusion and fixation
		6.3.3.2 Embedding
		6.3.3.3 Sectioning and poststaining
	6.3.4 Data analysis
	6.3.5 Troubleshooting
		6.3.5.1 Poststaining versus en bloc staining
		6.3.5.2 Osmolality, electrolytes, and additives in fixation
	6.3.6 Concluding remarks
	References
6.4 Transmission electron microscopy for tracer assay
	6.4.1 Background knowledge
		6.4.1.1 Tissue barrier defined by tracer
		6.4.1.2 Lanthanum
	6.4.2 Materials and reagents
		6.4.2.1 Equipment
		6.4.2.2 Buffers
	6.4.3 Experimental procedure
	6.4.4 Data analysis
	6.4.5 Troubleshooting
	6.4.6 Concluding remarks
	References
6.5 Transmission electron microscopy for immunolabeling application
	6.5.1 Background knowledge
		6.5.1.1 Immunoelectron microscopy
		6.5.1.2 Low temperature embedding
	6.5.2 Materials and reagents
		6.5.2.1 Equipment
		6.5.2.2 Buffers
	6.5.3 Experimental procedure
		6.5.3.1 Perfusion and fixation
		6.5.3.2 Embedding
		6.5.3.3 Sectioning and immunolabeling
	6.5.4 Data analysis
	6.5.5 Troubleshooting
		6.5.5.1 Negative result
		6.5.5.2 Nonspecific binding
	6.5.6 Concluding remarks
	References
6.6 Freeze-fracture electron microscopy
	6.6.1 Background knowledge
		6.6.1.1 Principle of freeze-fracture technique
		6.6.1.2 Technical consideration
			6.6.1.2.1 Freezing
			6.6.1.2.2 Fracturing
			6.6.1.2.3 Etching
			6.6.1.2.4 Replication
	6.6.2 Materials and reagents
		6.6.2.1 Equipment
		6.6.2.2 Cell model
		6.6.2.3 Cell culture medium
		6.6.2.4 Buffers
	6.6.3 Experimental procedure
		6.6.3.1a Cell culture and fixation
		6.6.3.1b Perfusion and fixation
		6.6.3.2 Freeze fracturing
	6.6.4 Data analysis
	6.6.5 Troubleshooting
	6.6.6 Concluding remarks
	References
6.7 Freeze-fracture replica immunolabeling technique
	6.7.1 Background knowledge
	6.7.2 Materials and reagents
		6.7.2.1 Equipment
		6.7.2.2 Cell model
		6.7.2.3 Cell culture medium
		6.7.2.4 Buffers
	6.7.3 Experimental procedure
	6.7.4 Data analysis
	6.7.5 Troubleshooting
	6.7.6 Concluding remarks
	References
7 Cell models of tight junction biology
7.1 Cell culture
	7.1.1 Primary culture and cell transformation
	7.1.2 Subculture and propagation
	7.1.3 Anchorage independence
	7.1.4 Cloning and selection
	7.1.5 Gene transfer
	7.1.6 Cryopreservation
	7.1.7 Contamination
	References
7.2 Culture of epithelial cells
	7.2.1 Background knowledge
		7.2.1.1 Epithelial phenotypes
		7.2.1.2 Epithelial cell lines
	7.2.2 Materials and reagents
		7.2.2.1 Equipment
		7.2.2.2 Cell model
		7.2.2.3 Cell culture medium
	7.2.3 Experimental procedure
		7.2.3.1 Growing MDCK cells on plastic substrate
		7.2.3.2 Seeding MDCK cells on permeable Transwell filter
	7.2.4 Data analysis
	7.2.5 Troubleshooting
	7.2.6 Concluding remarks
	References
7.3 Calcium switch assay
	7.3.1 Background knowledge
	7.3.2 Materials and reagents
		7.3.2.1 Equipment
		7.3.2.2 Cell model
		7.3.2.3 Cell culture medium
		7.3.2.4 Ca++-switch buffers
	7.3.3 Experimental procedure
	7.3.4 Data analysis
	7.3.5 Troubleshooting
	7.3.6 Concluding remarks
	References
7.4 Retrovirus-mediated transgene expression
	7.4.1 Background knowledge
		7.4.1.1 Retrovirus-mediated gene transfer
		7.4.1.2 Recombinant retroviral vector
		7.4.1.3 Packaging of retrovirus
		7.4.1.4 Pseudotyping with VSV-G protein
	7.4.2 Materials and reagents
		7.4.2.1 Cell model for ectopic gene expression
		7.4.2.2 Plasmids
		7.4.2.3 Cell culture medium
		7.4.2.4 Buffers
	7.4.3 Experimental procedure
	7.4.4 Data analysis
	7.4.5 Troubleshooting
		7.4.5.1 Low viral titer
		7.4.5.2 Low transduction efficiency
	7.4.6 Concluding remarks
	References
7.5 Retrovirus-mediated RNA interference
	7.5.1 Background knowledge
		7.5.1.1 Concept of RNA interference
		7.5.1.2 RNA interference as a tool to study loss of gene function
		7.5.1.3 Sequence selection for RNA interference
	7.5.2 Materials and reagents
		7.5.2.1 Cell model for ectopic gene expression
		7.5.2.2 Plasmids
		7.5.2.3 Cell culture medium
		7.5.2.4 Buffers
	7.5.3 Experimental procedure
	7.5.4 Data analysis
	7.5.5 Troubleshooting
		7.5.5.1 Expression level of siRNA
		7.5.5.2 Expression level of target gene
	7.5.6 Concluding remarks
	References
8 Mouse models of tight junction physiology
8.1 Mouse genetics and transgenics
	8.1.1 Laboratory mouse
	8.1.2 Mouse strain
		8.1.2.1 Inbred mouse strain
		8.1.2.2 Congenic mouse strain
		8.1.2.3 Hybrid mouse strain
		8.1.2.4 Outbred mouse strain
	8.1.3 Mouse genome
	8.1.4 Random mutagenesis in laboratory mouse
	8.1.5 Transgenesis in laboratory mouse
	8.1.6 Gene targeting in laboratory mouse
		8.1.6.1 Manipulation of mouse embryonic stem cells
		8.1.6.2 Homologous recombination
	References
8.2 Transgenic overexpression by DNA injection
	8.2.1 Background knowledge
		8.2.1.1 Transcription regulation
			8.2.1.1.1 General transcription machinery
			8.2.1.1.2 Core promoter architecture
		8.2.1.2 Transgene design
			8.2.1.2.1 Promoter and regulatory elements
			8.2.1.2.2 Intron–exon boundaries, Kozak sequence, and polyadenylation
		8.2.1.3 Pronuclear injection of mouse embryo
	8.2.2 Materials and reagents
		8.2.2.1 Equipment
		8.2.2.2 Buffers
	8.2.3 Experimental procedure
		8.2.3.1 Transgene release
		8.2.3.2 Pronuclear injection of DNA and production of transgenic mice
	8.2.4 Data analysis
	8.2.5 Troubleshooting
		8.2.5.1 Unwanted transgene expression
		8.2.5.2 Transgene silencing
		8.2.5.3 Transgenic mosaicism
	8.2.6 Concluding remarks
	References
8.3 Lentivirus-mediated gene knockdown
	8.3.1 Background knowledge
		8.3.1.1 Lentivirus-mediated transgenesis
		8.3.1.2 RNA interference in live mice
	8.3.2 Materials and reagents
		8.3.2.1 Equipment
		8.3.2.2 Cell model for ectopic gene expression
		8.3.2.3 Plasmids
		8.3.2.4 Cell culture medium
		8.3.2.5 Buffers
	8.3.3 Experimental procedure
		8.3.3.1 Lentivirus production
		8.3.3.2 Perivitelline injection of lentivirus and production of transgenic mice
	8.3.4 Data analysis
	8.3.5 Troubleshooting
		8.3.5.1 Silencing of recombinant lentivirus
		8.3.5.2 Toxicity of siRNA expression
		8.3.5.3 Knockdown versus knockout
	8.3.6 Concluding remarks
	References
8.4 Conditional gene knockout by homologous recombination
	8.4.1 Background knowledge
		8.4.1.1 Site-specific recombination system
		8.4.1.2 Design of targeting vector
	8.4.2 Materials and reagents
		8.4.2.1 Equipment
		8.4.2.2 Cell line
		8.4.2.3 Cell culture medium
		8.4.2.4 Buffer
	8.4.3 Experimental procedure
		8.4.3.1 Plating embryonic stem cells
		8.4.3.2 DNA electroporation
		8.4.3.3 Embryonic stem cell screening
		8.4.3.4 Embryonic stem cell injection to blastocyst
	8.4.4 Data analysis
		8.4.4.1 Screening of targeted embryonic stem cell clones
		8.4.4.2 Breeding strategy for mutant mice
	8.4.5 Troubleshooting
		8.4.5.1 Issues related to targeting vector
		8.4.5.2 Issues related to Cre expression
		8.4.5.3 Cell autonomy
	8.4.6 Concluding remarks
	References
9 Perspective
9.1 Scanning ion conductance microscopy
	9.1.1 Concept of conductance scanning
	9.1.2 Practical application
	9.1.3 Instrumentation
	9.1.4 Tight junction conductance measurement
	9.1.5 Limitation and future direction
	References
9.2 Cryo-electron microscopy
	9.2.1 Single-particle cryo-electron microscopy
		9.2.1.1 Structural determination without crystallization
		9.2.1.2 Protein quality and size
	9.2.2 Cryo-electron microscopy of vitreous section
	9.2.3 Limitation and future direction
	References
9.3 Super-resolution microscopy for tight junction
	9.3.1 Super-resolution microscopy
	9.3.2 Spatial separation of tight junction components
	9.3.3 Architectural alteration in tricellular tight junction
	9.3.4 Limitation and future direction
	References
9.4 Novel binders to tight junction
	9.4.1 Clostridium perfringens enterotoxin
	9.4.2 TJ modulating peptidomimetics
		9.4.2.1 Occludin peptidomimetics
		9.4.2.2 Claudin peptidomimetics
	9.4.3 Anti-claudin antibodies
	9.4.4 Limitation and future direction
	References
9.5 De novo assembly of tight junction
	9.5.1 Concept of de novo assembly of subcellular organelle
	9.5.2 Giant unilamellar vesicle
	9.5.3 Protein incorporation and topological orientation in giant unilamellar vesicle
	9.5.4 Probing claudin interactions in giant unilamellar vesicle
	9.5.5 Limitation and future direction
	References
9.6 Organoid model of tight junction biology
	9.6.1 Organoid culture
	9.6.2 Organ on a chip
	9.6.3 Bioprinting of organ
	9.6.4 Limitation and future direction
	References
Index