1 Useful Tips, Widely Used Techniques, and Quantifying Cell Metabolic Behavior
1.2 Baculovirus Infection
2.2 Baculovirus Infection
3.1.1 Cell Density and Viability
3.1.2 Evaluating μ, PDT, Lag Time, and Maximum Cell Density
3.1.3 Cell Growth: Useful Tips
3.2 Baculovirus Infection
3.2.1 Recombinant Baculovirus Development
3.2.2 Baculovirus Amplification
3.2.3 Determining Baculovirus Titer
3.2.4 Poisson Distribution
3.2.5 Infection of Insect Cell Cultures: Cell Line Selection
3.2.6 Infection of Insect Cell Cultures: Time of Infection and MOI
3.2.7 Infection of Insect Cell Cultures: Product Harvest Time
3.2.8 Baculovirus Infection: Useful Tips
3.3.1 Specific Oxygen Utilization Rate: Using a Dissolved Oxygen (DO) Electrode
3.3.2 Specific Oxygen Utilization Rate: Using a Dissolved Oxygen (DO) Monitor
3.3.3 Specific Nutrient Utilization and Byproduct Accumulation Rates
3.3.4 Specific Rates During Exponential Growth Phase
3.3.5 Specific Rates During Constant Cell Density
Part II: Baculovirus Molecular Biology/Development of Recombinant Baculoviruses
2 Introduction to Baculovirus Molecular Biology
4 Baculovirus Replication In Vivo
5 Baculovirus Gene Expression and Replication
5.1 Early Gene Expression
5.1.2 Transcriptional Enhancers
5.1.3 Transactivational Regulators
5.2 Baculovirus and Apoptosis
5.3 Baculovirus Replication
5.4 Late and Very Late Gene Expression
5.4.1 Viral RNA Polymerase
5.4.3 Regulation of Late and Very Late Gene Expression
6 Baculoviruses as Expression Vectors
3 Baculovirus Transfer Vectors
1.1 The Role of the Baculovirus Transfer Vector
1.2 The Parental Baculovirus Genome
2 Transfer Vectors for Expression of Single Genes
3 Transfer Vectors for Multiple Gene Expression
3.1 Polyhedrin Gene Locus
4 Transfer Vectors for Secretion of Recombinant Proteins or Surface Display on the Virus
5 Ligation Independent Cloning of Genes Into Transfer Vectors
4 Recombinant Baculovirus Isolation
2.1 Co-transfection of Insect Cells with Virus DNA and Transfer Vector DNA
2.2 Plaque-Assay to Separate Recombinant Virus from Parental Virus
2.3 PlaqueưPurification of Recombinant Virus
2.4 Amplification of Recombinant Virus (Small-Scale)
2.5 Titration of Virus by Plaque-Assay
2.6 Titration of Virus by Quantitative PCR
2.7 Transformation of E. coli DH10Bac™ with pFastBac Vectors to Produce Recombinant Bacmid (Bac-toưBacđ System)
2.8 Isolation of Recombinant Bacmid DNA for Transfection into Insect Cells (Bac-to-Bacđ System)
3.1 Co-transfection of Insect Cells with Virus DNA and Transfer Vector DNA
3.2 Plaque Assay to Separate Recombinant Virus from Parental Virus
3.3 PlaqueưPurification of Recombinant Virus
3.4 Amplification of Recombinant Virus
3.5 Plaque Assay to Titrate Amplified Virus
3.6 Titration of Virus by Quantitative PCR
3.7 Transformation of E. coli DH10Bac™ with pFastBac Vectors to Produce Recombinant Bacmid (Bac-toưBacđ System)
3.8 Isolation of Recombinant Bacmid DNA for Transfection into Insect Cells (Bac-to-Bacđ System)
5 Gene Expression in Mammalian Cells Using BacMam, a Modified Baculovirus System
3.1 BacMam Transfer Vector Construction
3.1.6 Subcloning Using Restriction Enzymes
3.2 Generation of Bacmid DNA
3.2.1 Transformation of DH10Bac
3.2.2 Preparation of Bacmid DNA
3.3 Generation of BacMam Virus
3.3.2 Transfection of Sf-9 Cells
3.3.3 Harvesting the P0 Virus
3.3.4 Generating P1 Virus
3.3.5 Large-Scale Amplification of BacMams
3.4 BacMam Virus Titration
3.5 Long-Term Storage of BacMam Virus
3.7 Application of BacMams
3.7.1 Identification of Receptive Mammalian Cell Lines Using a BacMam GFP Transduction Control
3.7.2 Transduction of Adherent Mammalian Cells
3.7.3 Transduction of Mammalian Cells in Suspension
Part III: Insect Cell Culture
6 Available Lepidopteran Insect Cell Lines
7 Lepidopteran Insect Cell Line Isolation from Insect Tissue
3.3.3 Tracheals/ Tracheoles
3.3.8 Reproductive Organs
3.4 Initiation of Primary Cultures
3.7 Additional Subcultivations
3.7.1 Cultures in a Flask
3.7.2 Cultures in a Petri Dish
8 Development of Serum-Free Media for Lepidopteran Insect Cell Lines
1.2 Low-Cost SerumưFree Media
1.3 Chemically Defined Media
1.5 Feed Development Challenges
2.1 Cell Culture Equipment
2.1.1 Cell Culture (Suspension Cell Culture)
2.1.2 Cell Counts/Sampling
2.2 Cell Cryopreservation Equipment
2.3 Serum-Free Medium/Feed Preparation Equipment
2.4 Serum-Free Medium/Feed Preparation Requirements
2.6 Commercial Serum-Free Media (Selection Only)
2.7 Stock Solutions for a Conventional Serum-Free Feed (Chan’s Feed)
3.1 Serum-Free Media Preparation
3.1.1 Preparing 10 L Commercial Serum-Free Medium from a Powder Formulation
3.1.2 Preparing a Lipid Emulsion (80ì)
3.2 Serum-Free Feed Preparation
3.2.1 Preparing a Conventional Serum-Free Feed (Chan’s Feed)
3.3 General Cell Culture Techniques
3.3.1 Depyrogenation of Labware
3.3.2 Aseptic Technique in the Biological Safety Cabinet (BSC)
3.3.3 Filter-Sterilize Solutions (Small Scale, Up to 50 mL)
3.3.4 Filter-Sterilize Solutions (Large Scale, Up to 50 L)
3.3.5 Batch Suspension Cell Culture
3.3.6 Fed-Batch Suspension Cell Culture: Conventional Feed
3.3.7 Cell Density and Cell Viability Enumeration
3.3.8 Cell Cryopreservation: Freezing
3.3.9 Cell Cryopreservation: Thawing
9 Routine Maintenance and Storage of Lepidopteran Insect Cell Lines and Baculoviruses
2.2 Quality-Control/Storage
3.1 Stock Cell Cultures: Passage Methods
3.1.1 Preparation of Hood, Examination of Culture, and Labeling of Flask(s)
3.1.2 Suspension and Loosely Attached Cell Lines
3.1.3 Moderately Attached Cells
3.1.4 Strongly Attached Cells: Trypsinization to Dislodge Cells
3.1.5 Strongly Attached: Cell Transfer Following Trypsinization
3.2 Quality Control and Testing
3.2.2 Identification: Introduction
3.2.3 Identification: Isozymes
3.2.4 Identification: DNA Amplification Fingerprinting
Part IV: Protein Production with Recombinant Baculoviruses
10 Small-Scale Production of Recombinant Proteins Using the Baculovirus Expression Vector System
2.1 Cell Culture and Baculovirus
2.2 Titering Virus by End-Point Dilution Assay
2.3 Optimization of Infection Parameters in 24-Well Plate
2.4 Protein Production in Six-Well Plate
2.5 Protein Production in 50 mL Suspension Culture
3.1 Titrating Virus Stocks with the EndưPoint Dilution Assay
3.2 Optimization of Infection Parameters in 24-Well Plate
3.3 Protein Production in Six-Well Plate
3.4 Protein Production in 50 mL Suspension Culture
3.5 Sample Preparation for Protein Analysis
3.5.2 Lysis in SDS-PAGE Sample Buffer
11 Recombinant Protein Production in Large-Scale Agitated Bioreactors Using the Baculovirus Expression Vector System
2.2 Baculovirus Amplification and Quantification
2.3 Cell Growth and Recombinant Protein Production in an Agitated Bioreactor
3.1.1 Insect Cell Growth for Seeding Bioreactor
3.2 Baculovirus Amplification and Quantification
3.2.1 Virus Stock Amplification
3.2.2 Baculovirus Quantification
3.3 Large-Scale Recombinant Protein Production in Bioreactors
3.3.1 Bioreactor Sterilization
3.3.2 Bioreactor Inoculation and Protein Production
3.3.5 Dissolved Oxygen (DO)
3.3.9 Further Advanced On-Line Monitoring and Control of Bioreactor Cultures
12 Protein Expression in Insect and Mammalian Cells Using Baculoviruses in Wave Bioreactors
3.1 Preparation of Cell Inoculum
3.2 Large-Scale Baculovirus Amplification: P2 Stocks
3.3 Setup of the Wave Cellbag, Insect Cells, and Mammalian Cells
3.3.1 Setup of Wave System200
3.3.2 Setup of Wave System20/50
3.4 Media Addition to the Bioreactor
3.5 Cell Addition to the Bioreactor
3.5.1 Sf-9 Cells for Insect Cell Production Run
3.5.2 CHO-GE Cells for Mammalian Cell Production Run
3.5.3 Cell Addition and Monitoring, Both Cell Types
3.6.1 Infection: Insect Cell Protein Production
3.6.2 Transduction: Mammalian Cell Protein Production
3.6.3 Virus Addition to Cellbag, Both Cell Types
3.7 Monitoring During Production Run
3.8.1 Cell Harvest, Insect or Mammalian, for General Protein Purification Use
3.8.2 Secreted Protein (Supernatant) Harvest, Insect or Mammalian Protein Production Runs: Low Endotoxin Conditions
13 Protein Production with Recombinant Baculoviruses in Lepidopteran Larvae
2.1 Rearing of Trichoplusia ni Larvae
2.2 Inoculation by Injection
2.4 Transfection of Larvae with Bacmid DNA
2.5 Homogenization and Clarification
3.3 Trichoplusia ni Rearing
3.3.1 Larval Rearing Starting with Eggs on a Substrate
3.3.2 Larval Rearing Starting with Loose Eggs
3.4 Determination of Developmental Stage
3.6.3 Larval Transfection
3.7 Incubation and Harvest Time
3.8 Homogenization, Clarification, Extraction and Purification
14 Production of Virus-Like Particles for Vaccination
2.1 Cell Lines and Recombinant Viruses
2.3 Shake Flask and Bioreactor Culture
2.5.3 Total Protein Analysis
2.5.4 Electron Microscopy
3.2 Baculovirus Stock Amplification
3.3 VLP and Vector Production
3.3.1 Bioreactor Preparation
3.3.3 Culture Harvest and VLP Extraction
3.3.5 VLP Downstream Processing
3.4.1 SDS PAGE/Western Analysis
3.4.3 Negative Staining Electron Microscopy
15 Alternative Strategies for Expressing Multicomponent Protein Complexes in Insect Cells
2.3 Automated Purification
3.1 Preparation of Baculovirus Infected Insect Cells (BIIC)
3.2 Expression of Multicomponent Protein Complexes in Sf-9 Cells with BIIC
3.4 KingFisher mL Automated Purification
3.7 Example 2CSN (COP9 Signalosome)
Part V: Recombinant Protein Production with Transformed Insect Cells
16 Transforming Lepidopteran Insect Cells for Continuous Recombinant Protein Expression
3.1 Insertion of the Gene of Interest into an Expression Plasmid
3.3 Selection and Isolation of Transformed Clones by the Colony Formation Method
3.4 Selection and Isolation of Transformed Clones by the Limiting Dilution Method
3.5 Screening for the Presence and Expression of the Gene of Interest
17 Stable Drosophila Cell Lines: An Alternative Approach to Exogenous Protein Expression
1.1 Limitations of the BEVS
1.2 Advantages of Expression in Drosophila S2 Cells
2.2 Medium and Cell Culture
3.2 Passaging of S2 Cells
3.4 Transfection and Selection of Stable Transfectants
3.4.1 Day 0 (Preparation for Transfection)
3.4.2 Day 1 (Transfection into S2 Cells Using Effectene)
3.4.3 Day 2 (Change of Medium)
3.4.4 Day 4 (Start of the Selection Process)
3.4.5 Day 9 (Change of Medium)
3.4.10 Day 28 (Expansion and Freezing Stocks of the Stable Cell Line)
3.4.6 Day 14 (Change of Medium)
3.4.7 Day 18 (Amplification and Adaptation to Serum-Free Medium)
3.4.8 Day 21 (Expansion and Adaptation to Serum-Free Medium)
3.5 1 L Culture Production
3.5.3 Day 12 (Production)
3.5.4 Day 15 (Production; Can Be Extended up to Day 18)
3.5.5 Day 22 (Production)
18 Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression
3.1 Transformation, Selection, and Isolation of Transformed Clones
3.2 Screening for Integration and Expression of New Protein Processing Gene(s)
3.3 Assessing Growth Properties of Stably Transformed Cells
3.4 Assessing the Susceptibility of Stably Transformed Cells to Baculovirus Infection
3.5 Assessing BaculovirusưMediated Foreign Gene Expression in Stably Transformed Cells
3.6 Assessing Protein Processing by Transformed Cells
3.7 Examples of Stable Transformation for Improved Protein Processing
3.7.1 Sf-9 and Sf-21 Cells Transformed for Altered Protein N-Glycosylation
3.7.2 Sf-9 and Tn-5 Cells Transformed for Improved Protein Folding
3.7.3 Sf-9 Cells Transformed for Increased Recombinant Protein Expression
Part VI: Baculovirus Development and Production for Use as Insecticides
19 Introduction to the Use of Baculoviruses as Biological Insecticides
2 Commercial Use of Baculovirus Insecticides
3 Pros and Cons of Baculovirus Insecticide Use
4 Genetic Enhancement of Baculovirus Insecticides
4.2 Ecdysteroid UDP-Glucosyltransferase
4.4 Field Testing of Recombinant Viruses
20 Baculovirus Insecticide Production in Insect Larvae
2.1 Rearing of H. zea Larvae and Determination of Larval Instar
2.2 Occlusion Body Concentration Determination by Hemacytometer Count
2.3 Larval Harvesting and Downstream Processing
2.4 Production of Recombinant AcMNPV in T. ni Larvae
3.1 Production of Wild Type HzNPV in H. zea Larvae
3.2 Rearing of H. zea Larvae and Determination of Larval Instar
3.3 Occlusion Body Concentration Determination by Hemacytometer Count
3.5 Larval Harvesting and Downstream Processing of HzNPV OBs
3.6 Production of Recombinant AcMNPV in T. ni Larvae
3.7 Larval Harvesting and Downstream Processing of AcNPV-ưLqhIT2 OBs
21 Evaluation of the Insecticidal Efficacy of Wild Type and Recombinant Baculoviruses
2.3 Insect Rearing Supplies
2.4 Supplies for Preparation of Polyhedra (Occlusion Bodies)
2.5.1 Droplet Feeding Method
2.5.2 Diet Plug Feed Method
2.5.3 Microinjection Method
2.5.4 Feeding Damage Method
2.5.5 Statistical Packages
3.2 Preparation of Polyhedra for Bioassay
3.2.1 Larval Propagation of Polyhedra
3.2.2 Cell Culture Propagation of Polyhedra
3.2.3 Quantification of Polyhedra
3.3 Droplet Feeding Bioassay for LC50 Determination
3.3.1 Calculation of Range of Concentrations
3.3.2 Infection by Droplet Feeding
3.4 Diet Plug Bioassay For Determination of Median Lethal Dose
3.5 Microapplicator-Assisted Bioassays Using Budded Virus
3.6 Droplet Feeding for ST50 Determinations
3.8.1 LC50 or LD50 Determination: The Probit Analysis
3.8.2 Comparing of the LC50 of Two or More Baculoviruses
3.8.3 ST50 Calculation Using Life Testing
3.8.4 Estimating LC50 Using Time of Inspection and Concentration Simultaneously
Part VII: Miscellaneous Techniques and Applications of the Baculovirus/Insect Cell System
22 Evaluating Baculovirus Infection Using Green Fluorescent Protein and Variants
2.1 Cell Culture and Medium
2.2 Expression Vector and Baculovirus
2.3 Western Blotting for GFP
3.1 Construction of Recombinant Expression Vectors and Establishment of Recombinant Baculoviruses
3.2 End-Point Dilution and Titer Determination Using GFP
3.3 Early Monitoring of Viral Infection Using ETL-GFP Strategy
23 Tubular Bioreactor for Probing Baculovirus Infection and Protein Production
2.1 Cell Culture, Medium, and Assays
2.2 Two-Stage Bioreactor System
24 Gene Silencing in Insect Cells Using RNAi
2.2 Making the DNA Template
2.3 In Vitro dsRNA Synthesis
2.4 Transfecting Sf-21 Cells in Culture
3.2 Making the Template DNA
3.3 In Vitro dsRNA Synthesis
3.4 Transfecting Sf-21 Cells in Culture
25 Using the Baculovirus/Insect Cell System to Study Apoptosis
2.1 Chemicals and Solutions
3.1 Utilization of a BaculovirusưBased System to Discover Genes that Inhibit Apoptosis
3.1.1 Infection of Insect Cells with Baculoviruses
3.1.2 Isolation of AcMNPV DNA
3.1.3 Transfection Mixture Preparation
3.1.5 Induction of Apoptosis in Insect Cells by Baculovirus Infection
3.1.6 Induction of Apoptosis in Insect Cells by Transfection
3.1.7 Monitoring Apoptosis by Direct Microscopic Observation of the Blebbing Cells
3.1.8 Monitoring Apoptosis by Extraction of Fragmented DNA (Oligonucleosomes)
3.1.9 Genetic Screen and Rescue of AcMNPV Replication
3.1.10 Identification of the ApoptosisưSuppressing Gene
3.1.11 Isolation of Recombinant Baculoviruses
3.2 Utilization of BaculovirusưBased Plasmids to Extend the Applicability of the Assay to Discover Anti-apoptosis Genes in Vertebrates and Invertebrates
3.3 Evaluating Structural Motifs of ApoptosisưSuppressing Proteins
3.3.1 Site-Directed Mutagenesis Using Overlap Extension Polymerase Chain Reaction
3.3.2 Quantitative Evaluation of the Marker Rescue Assay
26 Generation of Envelope-Modified Baculoviruses for Gene Delivery into Mammalian Cells
1.1 Principle of Baculovirus Envelope Modification
1.2 Selection of the Envelope Sequence Based on Intended Use
1.2.1 Complement-Resistant Vectors
2.1 Transfer Plasmid for Envelope Modification and Mammalian Gene Expression
2.4 Titration by Plaque Assay
2.5 Amplification of Recombinant Viruses
2.6 Virus Concentration and Purification
2.7.2 Electron Microscopy
3.1 Transfer Plasmid for Envelope Modification and Mammalian Gene Expression
3.1.1 Basic Vector Elements
3.1.2 Insertion of a Mammalian Expression Cassette
3.1.3 PCR-Cloning of New Envelope Sequence
3.1.4 Baculovirus Genomic DNA
3.2 Insect Cell Culture, Serum-Free Versus Serum Containing
3.3.1 Producing the Recombinant Virus by Homologous Recombination in Insect Cells
3.3.2 Isolation of Single Recombinant Viruses, Titration, and Storage
3.3.3 Amplification of Recombinant Viruses
3.3.4 Virus Concentration
3.3.5 Virus Purification (See Note 6)
3.4.3 Electron Microscopy
3.5 Gene Delivery into Mammalian Cells
3.5.1 Transduction Procedure
3.5.2 Analysis of Gene Delivery
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