An Introduction to Molecular Biotechnology: Fundamentals, Methods and Applications

Completely updated in line with the rapid progress made in the field, this new edition of the highly-praised textbook addresses powerful new methods and concepts in biotechnology, such as genome editing, reprogrammed stem cells, and personalized medicine. An introduction to the fundamentals in molecular and cell biology is followed by a description of standard techniques, including purification and analysis of biomolecules, cloning techniques, gene expression systems, genome editing methods, labeling of proteins and in situ-techniques, standard and high resolution microscopy. The third part focuses on key areas in research and application, ranging from functional genomics, proteomics and bioinformatics to drug targeting, recombinant antibodies and systems biology. The final part looks at the biotechnology industry, explaining intellectual property issues, legal frameworks for pharmaceutical products and the interplay between start-up and larger companies. The contents are beautifully illustrated throughout, with hundreds of full color diagrams and photographs. Provides students and professionals in life sciences, pharmacy and biochemistry with everything they need to know about molecular biotechnology.

Table of Contents:
Abbreviations xix Part I Fundamentals of Cellular and Molecular Biology 1 1 The Cell as the Basic Unit of Life 3 Michael Wink References 8 Further Reading 8 2 Structure and Function of Cellular Macromolecules 9 Michael Wink 2.1 Structure and Function of Sugars 9 2.2 Structure of Membrane Lipids 13 2.3 Structure and Function of Proteins 17 2.4 Structure of Nucleotides and Nucleic Acids (DNA and RNA) 25 References 32 Further Reading 32 3 Structure and Functions of a Cell 33 Michael Wink 3.1 Structure of a Eukaryotic Cell 33 3.1.1 Structure and Function of the Cytoplasmic Membrane 33 3.1.1.1 Membrane Permeability 33 3.1.1.2 Transport Processes Across Biomembranes 34 3.1.1.3 Receptors and Signal Transduction at Biomembranes 37 3.1.2 Endomembrane System in a Eukaryotic Cell 40 3.1.3 Mitochondria and Chloroplasts 45 3.1.4 Cytoplasm 49 3.1.5 Cytoskeleton 51 3.1.6 Cell Walls 53 3.2 Structure of Bacteria 53 3.3 Structure of Viruses 55 3.4 Differentiation of Cells 56 3.5 Cell Death 60 References 61 Further Reading 61 4 Biosynthesis and Function of Macromolecules (DNA, RNA, and Proteins) 63 Michael Wink 4.1 Genomes, Chromosomes, and Replication 63 4.1.1 Genome Size 63 4.1.2 Composition and Function of Chromosomes 67 4.1.3 Mitosis and Meiosis 69 4.1.4 Replication 71 4.1.5 Mutations and Repair Mechanisms 72 4.2 Transcription: From Gene to Protein 77 4.3 Protein Biosynthesis (Translation) 81 Further Reading 85 5 Distributing Proteins in the Cell (Protein Sorting) 87 Michael Wink 5.1 Import and Export of Proteins via the Nuclear Pore 87 5.2 Import of Proteins in Mitochondria, Chloroplasts, and Peroxisomes 88 5.3 Protein Transport into the Endoplasmic Reticulum 89 5.4 Vesicle Transport from the ER via the Golgi Apparatus to the Cytoplasmic Membrane 92 References 94 Further Reading 94 6 Evolution and Diversity of Organisms 95 Michael Wink 6.1 Prokaryotes 95 6.2 Eukaryotes 95 References 101 Further Reading 101 Part II Standard Methods in Molecular Biotechnology 103 7 Isolation and Purification of Proteins 105 Thomas Wieland 7.1 Introduction 105 7.2 Producing a Protein Extract 106 7.3 Gel Electrophoretic Separation Methods 107 7.3.1 Principles of Electrophoresis 107 7.3.2 Native Gel Electrophoresis 107 7.3.3 Discontinuous Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 107 7.3.4 Two-Dimensional (2D) Gel Electrophoresis and Isoelectric Focusing (IEF) 108 7.3.5 Detecting Proteins in Gels 108 7.4 Methods of Protein Precipitation 109 7.5 Column Chromatography Methods 109 7.5.1 General Principles of Separation 109 7.5.1.1 Size Exclusion Chromatography (Gel Filtration) 109 7.5.1.2 Hydrophobic Interaction Chromatography 111 7.5.1.3 Ion Exchange Chromatography 111 7.5.1.4 Hydroxyapatite Chromatography 112 7.5.2 Group-Specific Separation Techniques 112 7.5.2.1 Chromatography on Protein A or Protein G 112 7.5.2.2 Chromatography on Cibacron Blue (Blue Gel) 112 7.5.2.3 Chromatography on Lectins 112 7.5.2.4 Chromatography on Heparin 113 7.5.3 Purification of Recombinant Fusion Proteins 113 7.5.3.1 Chromatography on Chelating Agents 113 7.5.3.2 Chromatography on Glutathione Matrices 114 7.6 Examples 114 7.6.1 Example 1: Purification of Nucleoside Diphosphate Kinase from the Cytosol of Bovine Retina Rod Cells 114 7.6.2 Example 2: Purification of Recombinant His6-RGS16 After Expression in E. coli 114 Further Reading 115 8 Mass Spectrometry and Applications in Proteomics and Microbial Identification 117 Andreas Schlosser and Wolf D. Lehmann 8.1 Principles of ESI and MALDI Mass Spectrometry 117 8.2 Instrumental Setup 118 8.3 Intact Protein Analysis 119 8.3.1 Protein Digestion 119 8.3.2 Peptide Fragmentation 119 8.3.3 Protein Identification with MS/MS Spectra 121 8.4 Protein and Proteome Quantification 121 8.4.1 Label-Free Quantification 121 8.4.2 Chemical Stable Isotope Labeling 121 8.4.3 Metabolic Stable Isotope Labeling 122 8.5 Protein–Protein Interaction Analysis 123 8.6 Analysis of Posttranslational Modifications 124 8.7 Microbial Identification and Resistance Detection 125 References 126 9 Isolation of DNA and RNA 129 Hans Weiher 9.1 Introduction 129 9.2 DNA Isolation 129 9.3 RNA Isolation 131 9.3.1 Enrichment of mRNA 131 Reference 131 10 Chromatography and Electrophoresis of Nucleic Acids 133 Hans Weiher 10.1 Introduction 133 10.2 Chromatographic Separation of Nucleic Acids 133 10.3 Electrophoresis 134 10.3.1 Agarose Gel Electrophoresis: Submarine Electrophoresis 134 10.3.2 Pulsed-Field Agarose Gel Electrophoresis 134 10.3.3 Polyacrylamide Gel Electrophoresis (PAGE) 135 Further Reading 135 11 Hybridization of Nucleic Acids 137 Hans Weiher 11.1 Significance of Base Pairing 137 11.2 Experimental Hybridization: Kinetic and Thermodynamic Control 137 11.3 Analytical Techniques 138 11.3.1 Clone Detection, Southern Blotting, Northern Blotting, and Gene Diagnosis 138 11.3.2 Systematic Gene Diagnosis and Expression Screening Based on Gene Arrays 139 11.3.3 In Situ Hybridization 139 References 140 Further Reading 140 12 Use of Enzymes in the Modification of Nucleic Acids 141 Ingrid Herr and MichaelWink 12.1 Restriction Enzymes (Restriction Endonucleases) 141 12.2 Ligases 142 12.3 Methyl transferases 142 12.4 DNA Polymerases 143 12.5 RNA Polymerases and Reverse Transcriptase 144 12.6 Nucleases 144 12.7 T4 Polynucleotide Kinase 144 12.8 Phosphatases 145 Further Reading 145 13 Polymerase Chain Reaction 147 Richard Jäger and Hans Weiher 13.1 Introduction 147 13.2 PCR Methods 147 13.2.1 Basic Principle 147 13.2.2 Primer Design and Hot Start PCR 148 13.2.3 Multiplex PCR 149 13.2.4 RT-PCR 149 13.2.5 Qualitative Analysis of the PCR Products 149 13.3 PCR as a Quantitative Method 149 13.3.1 PCR Phases and PCR Efficiency 149 13.3.2 Quantitative Real-Time PCR 150 13.3.3 Digital PCR 151 13.4 Areas of Application 151 13.4.1 Genome Analysis 151 13.4.2 Cloning Techniques 152 13.4.3 Gene Expression Studies 152 Further Reading 152 14 DNA Sequencing 153 Richard Jäger and HansWeiher 14.1 Introduction 153 14.2 The Sanger Method 153 14.3 Pyrosequencing 154 14.4 Second-Generation Sequencing: Illumina and Ion Torrent 155 14.4.1 Overview 155 14.4.2 The Illumina Sequencing System 155 14.4.3 The Ion Torrent Sequencing System 156 14.5 Third-Generation Sequencing Techniques 156 14.5.1 Overview 156 14.5.2 SMRT Sequencing 157 14.5.3 Nanopore Sequencing 157 14.6 The Impact of the DNA Sequencing Technology 158 References 158 Further Reading 158 Websites 158 15 Cloning Procedures 159 Thomas Wieland and Susanne Lutz 15.1 Introduction 159 15.2 Construction of Recombinant Vectors 159 15.2.1 Insert 159 15.2.2 Vector 161 15.2.3 Essential Components of Vectors 162 15.2.3.1 Bacterial Origin of Replication (ori) 162 15.2.3.2 Antibiotic Resistance 162 15.2.3.3 Polylinkers 162 15.2.4 Cloning Using Recombination Systems 162 15.2.5 Further Components of Vectors for Prokaryotic Expression Systems 163 15.2.5.1 Promoter 163 15.2.5.2 Ribosome-Binding Site 163 15.2.5.3 Termination Sequence 164 15.2.5.4 Fusion Sequence 164 15.2.6 Further Components of Eukaryotic Expression Vectors 164 15.2.6.1 Eukaryotic Expression Vectors: Yeast 164 15.2.6.2 Eukaryotic Expression Vectors for Mammal Cells 165 15.2.6.3 Viral Expression Systems for Mammalian Cells 167 15.2.7 Nonviral Introduction of Heterologous DNA to Host Organisms (Transformation, Transfection) 168 15.2.7.1 Transformation of Prokaryotes 168 15.2.7.2 Transformation of Yeast Cells 169 15.2.7.3 Transfection of Mammal Cells 169 Further Reading 170 16 Expression of Recombinant Proteins 171 Thomas Wieland 16.1 Introduction 171 16.2 Expression of Recombinant Proteins in Host Organisms 171 16.2.1 Expression in E. coli 172 16.2.2 Expression in Yeasts 175 16.2.3 Expression in Insect Cells 177 16.2.3.1 Expression Based on Recombinant Baculoviruses 177 16.2.3.2 Expression of Proteins in Stably Transfected Insect Cells 178 16.2.4 Expression of Proteins in Mammalian Cells 178 16.3 Expression in Cell-Free Systems 179 16.3.1 Expression of Proteins in Reticulocyte Lysates 180 16.3.2 Protein Expression Using E. coli Extracts 180 Further Reading 180 17 Patch Clamp Method 181 Robert Kraft 17.1 Ion Channels 181 17.2 Technical Requirements of the Patch Clamp Method 181 17.3 Patch Clamp Configurations 182 17.4 Applications of the Patch Clamp Method 183 Reference 185 Further Reading 185 18 Cell Cycle Analysis 187 Stefan Wölfl 18.1 Introduction 187 18.2 Analyzing the Cell Cycle 187 18.3 Experimental Analysis of the Cell Cycle 189 18.3.1 Preparing Synchronized Cell Cultures of S. cerevisiae 189 18.3.1.1 Centrifugal Elutriation 190 18.3.1.2 Cell Cycle Arrest Using α-Factor 190 18.3.2 Identification of Cell Cycle Stages 191 18.3.2.1 Budding Index 191 18.3.2.2 Fluorescent Staining of the Nucleus 191 18.3.2.3 Detection of Cell Cycle Phases Using Fluorescent Proteins as Reporters 194 Acknowledgments 195 Further Reading 196 19 Microscopic Techniques 197 Stephan Diekmann 19.1 Introduction 197 19.2 Electron Microscopy 197 19.2.1 Cryo-electron Microscopy 199 19.2.2 Electron Tomography 199 19.3 Atomic or Scanning Force Microscopy 199 19.3.1 Force Spectroscopy 200 19.3.2 Advantages and Disadvantages 201 19.4 Light Microscopy 201 19.4.1 Deconvolution 202 19.4.2 Confocal Microscopy 202 19.4.3 Why Fluorescence? 203 19.4.4 Nanoscopy 203 19.5 Microscopy in the Living Cell 204 19.5.1 Analysis of Fluorescently Labeled Proteins In Vivo 205 19.5.2 Fluorescence Recovery After Photobleaching 206 19.5.3 Fluorescence Correlation Spectroscopy 206 19.5.4 Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscopy 207 19.5.5 Single-Molecule Fluorescence 207 Further Reading 207 20 Laser Applications 209 Rainer Fink 20.1 Laser Development: A Historical Perspective 209 20.2 Types of Lasers and Setups 210 20.3 Properties of Laser Radiation 210 20.4 Applications 211 20.4.1 Laser Scanning Microscopy 211 20.4.2 Optical Tweezers 212 20.4.3 Laser Microdissection and Laser Therapy 212 20.4.4 Manufacturing of Products in Medical Technology and Biotechnology Products 213 Further Reading 213 Part III Key Topics 215 21 Sequencing the Universe of Life 217 Stefan Wiemann 21.1 What to Sequence? 217 21.1.1 Whole-Genome Sequencing 217 21.1.2 Exome Sequencing 220 21.1.3 (Gene) Panel Sequencing 220 21.1.4 RNA Sequencing 221 21.1.4.1 Tag- vs. Full-Length Sequencing 221 21.1.4.2 Sequencing of RNA Species and Modifications 221 21.1.4.3 Sequencing of Single Cells 222 21.1.4.4 In Situ Sequencing 222 21.1.5 (Whole-Genome) Bisulfite Sequencing of DNA 223 21.1.6 Sequencing to Characterize Chromatin Structure and Beyond 223 21.2 Sequencing Projects: Human 224 21.2.1 Initial Sequencing of the Human Genome 224 21.2.2 The 1000 Genomes Project: Assessing Natural Variation 224 21.2.3 Screening for Genetic Disease 225 21.2.4 Sequencing of Populations 226 21.2.5 TCGA and ICGC: Screening for Cancer Driver Mutations 226 21.3 Sequencing Other Species, Environments,… 228 21.4 Sequencing in the Clinics: Personalizing Oncology 228 21.5 Sequencing in the Private Sector: Direct to Consumer Testing (DTC) 231 21.6 The Information Content of a Genome Sequence and Ethical Consequences 231 References 232 22 Cellular Systems Biology 239 Melanie Boerries, Hauke Busch, and Rainer König 22.1 Introduction 239 22.2 Analysis of Cellular Networks by Top-Down Approaches 240 22.2.1 Motivation 240 22.2.2 Definitions and Construction of the Networks 240 22.2.3 Gene Set Enrichment Tests 241 22.2.4 Inferring Gene Regulators Employing Gene Regulatory Models 242 22.2.5 Network Descriptors 243 22.2.5.1 Scale-Free Networks 243 22.2.5.2 Centrality 243 22.2.5.3 The Clustering Coefficient 244 22.2.6 Detecting Essential Enzymes with a Machine Learning Approach 244 22.2.7 Elementary Flux Modes 244 22.3 Overview over Bottom-Up Modeling of Biochemical Networks 247 22.3.1 Motivation 247 22.3.2 Choosing Model Complexity and Model Building 248 22.3.3 Model Simulation 251 22.3.4 Model Calibration 252 22.3.5 Model Verification and Analysis 254 22.3.6 Examples 254 Further Reading 258 References 259 23 Protein–Protein and Protein–DNA Interactions 261 Peter Uetz and Ehmke Pohl 23.1 Protein–Protein Interactions 261 23.1.1 Classification and Specificity: Protein Domains 261 23.1.2 Protein Networks and Complexes 262 23.1.3 Structural Properties of Interacting Proteins 262 23.1.4 Which Forces Mediate Protein–Protein Interactions? 263 23.1.4.1 Thermodynamics 264 23.1.4.2 Energetics 264 23.1.5 Methods to Examine Protein–Protein Interactions 264 23.1.6 Theoretical Prediction of Protein–Protein Interactions 266 23.1.7 Regulation of Protein–Protein Interactions 266 23.1.8 Biotechnological and Medical Applications of Protein–Protein Interactions 268 23.2 Protein–DNA Interactions 269 23.2.1 Specific Protein–DNA Interaction 269 23.2.2 Thermodynamic Consideration 270 23.2.3 Methods to Study Protein–DNA Interactions 270 23.2.3.1 Structural Classification of Protein–DNA Complexes 270 23.2.4 Regulatory Networks and System Biology 270 23.2.5 Medical Importance of Protein–DNA Interactions 273 23.2.6 Biotechnological Applications 274 References 275 Further Reading 275 24 Bioinformatics 277 Benedikt Brors 24.1 Introduction 277 24.2 Data Sources 277 24.2.1 Primary Databases: EMBL/GenBank/DDBJ, PIR, and Swiss-Prot 277 24.2.2 Genome Databases: Ensembl and GoldenPath 278 24.2.3 Motif Databases: BLOCKS, PROSITE, Pfam, ProDom, and SMART 278 24.2.4 Molecular Structure Databases: PDB and SCOP 278 24.2.5 Transcriptome Databases: SAGE, ArrayExpress, and GEO 279 24.2.6 Reference Databases: PubMed, OMIM, and GeneCards 279 24.2.7 Pathway Databases and Gene Ontology 279 24.3 Sequence Analysis 280 24.3.1 Kyte–Doolittle Plot, HelicalWheel Analysis, and Signal Sequence Analysis 280 24.3.2 Pairwise Alignment 281 24.3.2.1 Local/Global 281 24.3.2.2 Optimal/Heuristic 282 24.3.3 Alignment Statistics 282 24.3.4 Multiple Alignment 282 24.4 Evolutionary Bioinformatics 283 24.4.1 StatisticalModels of Evolution 283 24.4.2 Relation to Score Matrices 284 24.4.3 Phylogenetic Analysis 285 24.5 Gene Prediction 285 24.5.1 Neural Networks or HMMs Based on Hexanucleotide Composition 286 24.5.2 Comparison with Expressed Sequence Tags or Other Genomes (Fugu, Mouse) 286 24.6 Bioinformatics in Transcriptome and Proteome Analysis 287 24.6.1 Preprocessing and Normalization 287 24.6.2 Feature Selection 288 24.6.3 Similarity Measures: Euclidean Distance, Correlation, Manhattan Distance, Mahalanobis Distance, and Entropy Measures 288 24.6.4 Unsupervised Learning Procedures: Clustering, Principal Component Analysis, Multidimensional Scaling, and Correspondence Analysis 289 24.6.5 Supervised Learning Procedures: Linear Discriminant Analysis, Decision Trees, Support Vector Machines, and ANNs 289 24.6.6 Analysis of Overrepresentation of Functional Categories 290 24.7 Analysis of Ultraparallel Sequencing Data 291 24.7.1 Mapping of Ultraparallel Sequencing Data 291 24.7.2 Genome (Re-)sequencing 292 24.7.3 Transcriptome Sequencing 292 24.7.4 ChIP-seq 293 24.7.5 Epigenetic Analysis 293 24.7.6 Single-Cell Analysis 294 24.7.7 Bioethics of Human Sequencing Data 294 24.8 Bioinformatic Software 294 Further Reading 295 25 Drug Research 297 Manfred Koegl, Ralf Tolle, Ulrich Deuschle, Claus Kremoser, and Michael Wink 25.1 Introduction 297 25.2 Active Compounds and Their Targets 297 25.2.1 Identification of Potential Targets in the Human Genome 298 25.2.2 Comparative Genome Analysis 298 25.2.3 Experimental Target Identification: In Vitro Methods 299 25.2.4 Experimental Identification of Targets: Model Organisms 300 25.2.5 Experimental Target Identification in Humans 300 25.2.6 Difference Between Target Candidates and Genuine Targets 301 25.2.7 Biologicals 301 25.2.8 DNA and RNA in New Therapeutic Approaches 302 25.2.9 Patent Protection for Targets 303 25.2.10 Compound Libraries as a Source of Drug Discovery 304 25.2.11 High-Throughput Screening 304 25.2.12 High-Quality Paramounts in Screening Assays 304 25.2.13 Virtual Ligand Screening 306 25.2.14 Activity of Drugs Described in Terms of Efficacy and Potency 307 25.2.15 Chemical Optimization of Lead Structures 307 25.3 Preclinical Pharmacology and Toxicology 308 25.4 Clinical Development 309 25.5 Clinical Testing 309 Further Reading 310 26 Drug Targeting and Prodrugs 311 Gert Fricker 26.1 Drug Targeting 311 26.1.1 Passive Targeting by Exploiting Special Physiological Properties of the Target Tissue 311 26.1.2 Physical Targeting 312 26.1.3 Active Targeting 313 26.1.4 Cellular Carrier Systems 316 26.2 Prodrugs 316 26.2.1 Prodrugs to Improve Drug Solubility 316 26.2.2 Prodrugs to Increase Stability 317 26.3 Penetration of Drugs Through Biological Membranes 317 26.4 Prodrugs to Extend Duration of Effect 318 26.5 Prodrugs for the Targeted Release of a Drug 318 26.6 Prodrugs to Minimize Side Effects 320 References 320 27 Molecular Diagnostics in Medicine 323 Stefan Wölfl and Reinhard Gessner 27.1 Introduction 323 27.2 Uses of Molecular Diagnostics 323 27.2.1 Introduction 323 27.2.2 Monogenic and Polygenic Diseases 323 27.2.3 Individual Variability in the Genome: Forensics 325 27.2.4 Individual Variability in the Genome: HLA Typing 325 27.2.5 Individual Variability in the Genome: Pharmacogenomics 325 27.2.6 Individual Variability in the Genome: Susceptibility to Infectious Diseases 326 27.2.7 Viral Diagnosis 326 27.2.8 Microbial Diagnosis and Resistance Diagnosis 327 27.3 Which Molecular Variations Should be Detected 327 27.3.1 Point Mutations 327 27.3.2 Insertions and Deletions 328 27.3.3 Nucleotide Repeats 328 27.3.4 Deletion or Duplication of Genes 328 27.3.5 Recombination Between Chromosomes 329 27.3.6 Epigenetic Changes 329 27.4 Molecular Diagnostic Methods 330 27.4.1 DNA/RNA Purification 331 27.4.2 Detection of Target Sequence and Known Sequence Variations 331 27.4.2.1 Nucleic Acid Tests 331 27.4.2.2 Quantitative PCR 332 27.4.2.3 Multiplexing of Nucleic Acid Detection: Nucleic Acid Microarrays 333 27.4.2.4 Production and Manufacture of Microarrays 334 27.4.2.5 Applications of Fragment Length Analysis 335 27.4.2.6 Minisequencing 336 27.4.2.7 Determination of Unknown Mutations 336 27.5 Outlook 337 Further Reading 338 Historic Article: “News & Views” 338 Reviews 338 Web Link 338 Textbooks 338 28 Recombinant Antibodies and Phage Display 339 Gustavo Marçal Schmidt Garcia Moreira and Stefan Dübel 28.1 Introduction 339 28.2 Generation of Specific Recombinant Antibodies 340 28.2.1 Generation of Antibody Gene Libraries 341 28.2.2 Selection Systems for Recombinant Antibodies 342 28.2.2.1 Transgenic Mice with Human IgG Genes 342 28.2.2.2 In Vitro Selection Systems 342 28.3 Production and Purification of Recombinant Antibodies 348 28.4 Features and Applications of Recombinant Antibodies 349 28.4.1 Advantages of Recombinant Antibodies 349 28.4.2 Formats and Applications of Recombinant Antibodies 350 28.4.2.1 Camelid Antibodies and VH Domains 351 28.4.2.2 scFv and dsFv 351 28.4.2.3 scFv–Fc Fusions, Fc Engineering, and the Addition of Constant Domains 352 28.4.2.4 IgG, Fusion Proteins, and Derivatives for Therapy 352 28.4.2.5 Bispecific Antibodies 354 28.4.2.6 Chimeric Antigen Receptors (CARs) 355 28.4.3 The Future of Therapeutic Antibodies 355 28.4.4 Research and In Vitro Diagnostics 356 28.4.5 Intracellular and Cell-Penetrating Antibodies 356 28.5 Outlook 357 Further Reading 357 Textbooks 357 References 358 29 Genetically Modified Mice and Their Impact in Medical Research 361 Rolf Sprengel and Mazahir T. Hasan 29.1 Overview 361 29.2 Transgenic Mice 362 29.2.1 Retroviral Infection 362 29.2.2 Pronuclear Injection 363 29.3 Homologous Recombination: Knockout (Knock-In) Mice 364 29.4 Endonuclease-Based Knockout Mice 366 29.5 Endonuclease-Based Knock-In Mice 367 29.6 Conditionally Regulated Gene Expression 367 29.7 Gene Transfer to Subpopulations of Cells 368 29.7.1 Electroporation of Mouse Embryos (Plasmid DNA) 368 29.7.2 Virus-Mediated Gene Transfer (Lentivirus, rAAVs) 369 29.7.3 Virus-Mediated Gene Deletion (Cre/lox) 370 29.7.4 Virus-Mediated Gene Knockdown (shRNA, Antagomirs) 370 29.8 Impact of Genetically Modified Mice in Biomedicine 370 29.8.1 Alzheimer’s Disease 370 29.8.2 Amyotrophic Lateral Sclerosis (ALS) 370 29.8.3 Psychological and Cognitive Disorders 371 29.8.4 Autism Spectrum Disorder (ASD) 371 29.8.5 Chemogenetics, Optogenetics, and Magnetogenetics 372 29.9 Outlook 372 Reference 373 Further Reading 373 30 Plant Biotechnology 375 Helke Hillebrand and Rüdiger Hell 30.1 Introduction 375 30.1.1 Green Genetic Engineering: A New Method Toward Traditional Goals 375 30.1.2 Challenges in Plant Biotechnology 376 30.2 Gene Expression Control and Genome Editing 376 30.2.1 Gene Expression Control 377 30.2.2 Genome Editing 377 30.3 Production of Transgenic Plants 378 30.3.1 Transformation Systems 379 30.3.1.1 Agrobacterium as a Natural Transformation System 379 30.3.1.2 Biolistic Method: Gene Gun 381 30.3.1.3 Plastid Transformation 382 30.3.1.4 Viral Systems 382 30.4 Selection of Transformed Plant Cells 383 30.4.1 Requirements for an Optimal Selection Marker System 383 30.4.2 Negative Selection Marker Systems 384 30.4.3 Positive Selection Marker Systems 385 30.4.4 Selection Systems, Genetic Engineering Safety, and Marker-Free Plants 385 30.5 Regeneration of Transgenic Plants 387 30.5.1 Regeneration Procedures 387 30.5.2 Composition of Regeneration Media 387 30.6 Plant Analysis: Identification and Characterization of Genetically Engineered Plants 388 30.6.1 DNA and RNA Verification 388 30.6.2 Protein Analysis 389 30.6.3 Genetic and Molecular Maps 389 30.6.4 Stability of Transgenic Plants 390 Further Reading 390 31 Biocatalysis in the Chemical Industry 393 Michael Breuer and Bernhard Hauer 31.1 Introduction 393 31.2 Bioconversion/Enzymatic Procedures 395 31.3 Development of an Enzyme for Industrial Biocatalysis 397 31.3.1 Identification of Novel Biocatalysts 397 31.3.2 Improvement of Biocatalysts 399 31.3.3 Production of Biocatalysts 399 31.3.4 Outlook 399 31.3.5 Case Study 1: Screening for New Nitrilases 400 31.3.6 Case Study 2: Use of Known Enzymes for New Reactions: Lipases for the Production of Optically Active Amines and Alcohols 400 31.3.7 Case Study 3: Enzyme Optimization with Rational and Evolutive Methods 401 31.4 Fermentative Procedures 402 31.4.1 Improvement of Fermentation Processes 402 31.4.2 Classical Strain Optimization 403 31.4.3 Metabolic Engineering 404 31.4.4 Case Study 4: Fermentative Production of n-Butanol 405 31.4.5 Case Study 5: Production of Glutamic Acid with C. glutamicum 406 31.4.5.1 Molecular Mechanism of Glutamate Overproduction 406 31.4.6 Case Study 6: Production of Lysine with C. glutamicum 407 31.4.6.1 Molecular Mechanism of Lysine Biosynthesis 407 31.4.6.2 Deregulation of the Key Enzyme Aspartate Kinase 408 31.4.7 Genomic Research and Functional Genomics 409 31.4.8 Case Study 7: Fermentative Penicillin Production 409 31.4.9 Case Study 8: Vitamin B2 Production 409 31.4.9.1 Riboflavin Biosynthesis 410 31.4.9.2 Classical Strain Development 410 References 410 Part IV Biotechnology in Industry 411 32 Industrial Application: Biotech Industry,Markets, and Opportunities 413 Julia Schüler 32.1 Historical Overview and Definitions of Concepts 413 32.2 Areas of Industrial Application of Molecular Biotechnology 414 32.2.1 Red Biotechnology 414 32.2.1.1 Biopharmaceutical Drug Development 414 32.2.1.2 Gene and Cell Therapy 416 32.2.1.3 Tissue Engineering/Regenerative Medicine 419 32.2.1.4 Pharmacogenomics and Personalized Medicine 421 32.2.1.5 Molecular Diagnostic Agents 421 32.2.1.6 Systems Biology 422 32.2.1.7 Synthetic Biology 422 32.2.2 Green Biotechnology 422 32.2.2.1 Transgenic Plants 422 32.2.2.2 Genomic Approaches in Green Biotechnology 423 32.2.2.3 Novel Food and Functional Food 423 32.2.2.4 Livestock Breeding 423 32.2.3 White Biotechnology 424 32.3 Status Quo of the Biotech Industry Worldwide 424 32.3.1 Global Overview 424 32.3.2 United States 424 32.3.3 Europe 424 33 Patents in the Molecular Biotechnology Industry: Legal and Ethical Issues 425 David Resnik 33.1 Patent Law 425 33.1.1 What is a Patent? 425 33.1.2 How Does One Obtain a Patent? 426 33.1.3 What is the Proper Subject Matter for a Patent? 426 33.1.4 Types of Patents in Pharmaceutical and Molecular Biotechnology 427 33.1.5 Patent Infringement 427 33.1.6 International Patent Law 428 33.2 Ethical and Policy Issues in Biotechnology Patents 428 33.2.1 No Patents on Nature 428 33.2.2 Threats to Human Dignity 429 33.2.3 Problems with Access to Technology 430 33.2.4 Benefit Sharing 432 33.3 Conclusions 433 Acknowledgments 433 34 Drug Approval in the European Union and United States 435 Gary Walsh 34.1 Introduction 435 34.2 Regulation Within the European Union 435 34.2.1 The EU Regulatory Framework 435 34.2.2 The EMA and National Competent Authorities 436 34.2.3 New Drug Approval Routes 437 34.2.3.1 The Centralized Procedure 437 34.2.3.2 Decentralized Procedure and Mutual Recognition 438 34.3 Regulation in the United States 438 34.3.1 CDER and CBER 439 34.3.2 The Approvals Procedure 439 34.4 The Advent and Regulation of Biosimilars 440 34.5 International Regulatory Harmonization 441 References 442 35 Emergence of a Biotechnology Industry 445 Claus Kremoser Reference 451 Further Reading 451 36 The 101 of Founding a Biotech Company 453 Claus Kremoser and Michael Wink 36.1 First Steps Toward Your Own Company 453 36.2 Employees: Recruitment, Remuneration, and Participation 456 37 Marketing 459 Claus Kremoser and Michael Wink 37.1 Introduction 459 37.2 What Types of Deals Are Possible? 460 37.3 What Milestone or License Fees Are Effectively Paid in a Biotech/Pharma Cooperation? 460 37.4 PR and IR in Biotech Companies 461 Further Reading 462 Websites 462 Glossary 463 Index 491