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.
Michael Wink studied biology and chemistry in Bonn and was awarded his doctorate from TU Braunschweig in 1980. After gaining his lecturing qualification in 1984/1985, he was awarded a Heisenberg grant by the German Research Council to work at the Max Planck Institute for Breeding Research in Cologne and from then at the Gene Center of Ludwig-Maximilians University in Munich. Following a chair for Pharmaceutical Biology at Mainz University in 1988, he accepted the post of Professor for Pharmaceutical Biology at the University of Heidelberg one year later. His areas of interest include pharmaceutical research, molecular biotechnology, and medicinal plants, as well as research into natural products and evolution.
Abbreviations xix
Part I Fundamentals of Cellular and Molecular Biology1
1 The Cell as the Basic Unit of Life3
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 Cell33
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 Organisms95
Michael Wink
6.1 Prokaryotes 95
6.2 Eukaryotes 95
References 101
Further Reading 101
Part II Standard Methods in Molecular Biotechnology103
7 Isolation and Purification of Proteins105
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 inE. coli114
Further Reading 115
8 Mass Spectrometry and Applications in Proteomics and Microbial Identification117
Andreas Schlosser and Wolf D. Lehmann
8.1 Principles of ESI and MALDIMass 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 ProteinProtein 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 RNA129
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 Acids133
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 Acids137
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
11.3.3In SituHybridization 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 Reaction147
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 Sequencing153
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 Procedures159
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 Proteins171
Thomas Wieland
16.1 Introduction 171
16.2 Expression of Recombinant Proteins in Host Organisms 171
16.2.1 Expression inE. coli172
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 UsingE. coliExtracts 180
Further Reading 180
17 Patch Clamp Method181
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 Analysis187
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 -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 Techniques197
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 ProteinsIn Vivo205
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 Applications209
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 Topics215
21 Sequencing the Universe of Life217
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
21.1.4.4InSituSequencing 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 Biology239
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 ProteinProtein and ProteinDNA Interactions261
Peter Uetz and Ehmke Pohl
23.1 ProteinProtein 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 ProteinProtein Interactions? 263
23.1.4.1 Thermodynamics 264
23.1.4.2 Energetics 264
23.1.5 Methods to Examine ProteinProtein Interactions 264
23.1.6 Theoretical Prediction of ProteinProtein Interactions 266
23.1.7 Regulation of ProteinProtein Interactions 266
23.1.8 Biotechnological and Medical Applications of ProteinProtein Interactions 268
23.2 ProteinDNA Interactions 269
23.2.1 Specific ProteinDNA Interaction 269
23.2.2 Thermodynamic Consideration 270
23.2.3 Methods to Study ProteinDNA Interactions 270
23.2.3.1 Structural Classification of ProteinDNA Complexes 270
23.2.4 Regulatory Networks and System Biology 270
23.2.5 Medical Importance of ProteinDNA Interactions 273
23.2.6 Biotechnological Applications 274
References 275
Further Reading 275
24 Bioinformatics277
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 KyteDoolittle 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 Research297
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 VitroMethods 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 Prodrugs311
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 Medicine323
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 Display339
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
28.2.2.2In VitroSelection 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 scFvFc 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 andIn VitroDiagnostics 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 Research361
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 Alzheimers 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 Biotechnology375
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
30.3.1.1Agrobacteriumas 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 Industry393
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 ofn-Butanol 405
31.4.5 Case Study 5: Production of Glutamic Acid withC. glutamicum406
31.4.5.1 Molecular Mechanism of Glutamate Overproduction 406
31.4.6 Case Study 6: Production of Lysine withC. glutamicum407
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 Industry411
32 Industrial Application: Biotech Industry,Markets, and Opportunities413
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 States435
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 Industry445
Claus Kremoser
Reference 451
Further Reading 451
36 The 101 of Founding a Biotech Company453
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
