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An Introduction to Molecular Biotechnology. Fundamentals, Methods and Applications. Edition No. 3

  • Book
  • 544 Pages
  • February 2021
  • John Wiley and Sons Ltd
  • ID: 5185565
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

Authors

Michael Wink Universitat Heidelberg.