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Presentation of all the innovative methodologies in genome engineering
From basic science to practical applications
Concerns a broad range of organisms (from bacteria to mammals)
From basic science to practical applications
The post-genomic era has brought new challenges and opportunities in all fields of the biology. In this context, several genome engineering technologies have emerged that will help deciphering genes function by as well as improve gene therapy strategies. Genomic modifications such as knock-in, knock-out, knock-down, sequence replacement or modification can today be routinely performed. However, in front of this large palette of methodologies scientists may experience difficulties to gather useful information’s scattered within the literature. This book aims to present the state of this field from basic mechanisms of site-directed modifications to their applications in a wide range of organisms such as bacteria, yeast, plants, insects, mammals. It will discuss the problems encountered when using the random integration strategy and present the recent advances made in targeted genome modification. Technologies based on Zinc Finger nucleases, Meganucleases, TALEN, CRE and FLP recombinase, C31 integrase, transposases and resolvases are fully detailed with their strengths and weaknesses. All these information’s will help students and experienced researchers to understand and choose the best technology for their own purposes.
Preface.- Section 1: Site–directed integration of transgenes.- 1 Transgene site-specific integration : troubles and solutions.- 1.1 Introduction.- 1.2 Random integration and clonal dominance: reality or myth.- 1.3 Principal drawbacks of gene transfer integrative systems and solutions.- 1.4 Combining long-term expression and secure integration by specific locus targeting: old solutions to new perspectives.- 1.5 Conclusion.- 2 : Designing non-viral targetd integrating vectors for genome enginnering in vertebrates.- 2.1. Introduction : Ideal integrating vectors and safe insertion sites.- 2.2. Design of random integrating vectors based on DNA transposons and associated-targeting strategy.- 2. 3. Site-specific recombinase-based integrating vectors.- 2. 4. Meganuclease-based vectors.- Section 2: Integration based on homologous recombination.- 3: Gene targeting and homologous recombination in Saccharomyces cerevisiae.- 3.1 Introduction.- 3.2 Transformation with linearized plasmids: targeted integration.- 3.3 Mechanism of targeted integration: a state of the art of homologous recombination.- 3.4 Conclusions and future directions.- 4: Eucaryotic homologous recombination in mammalian cells.- 4.1. Models and products of HR.- 4.2. Molecular mechanisms of the different steps of HR.- 4.3. HR is at the heart of the genetic stability-diversity-instability equilibrium. - 4.4. The importance of sister chromatids in the cell cycle for the maintenance of genome stability.- 4.5. Deregulation of HR and tumor predisposition.- 4.6. HR in molecular evolution: concerted evolution.- 4.7. Comments/considerations regarding optimization of targeted gene replacement. – 5 Engineered Zinc Finger Nucleases for Targeted Genome Editing .- 5.1 Introduction.- 5.2 Zinc Fingers: Structure and Function.- 5.3 Zinc Finger Fusion Proteins.- 5.4 Zinc Finger Engineering Platforms.- 5.5 Modified ZFN Architectures.- 5.6 Types of ZFN-Inducible Genome Modifications.- 5.7 Cell Line and Organismic Modifications using ZFNs.- 5.8 Off-Target Effects and Cytotoxicity.- 5.9 Conclusions.- 6: Engineered meganucleases for genome engineering purposes.- 6.1. Introduction.- 6.2. Historical considerations.- 6.3. The I-SceI success story.- 6.4. The challenge of specificity toward a chosen sequence.- 6.5. Cleavage versus Nickase activity.- 6.6. Customized meganucleases for genome engineering.- 6.7. Overview on TAL Effector Nuclease (TALEN).-6.8. Conclusions.- Section3: Integration based on Site-specific recombination.- 7 : Cre/loxP, Flp/FRT systems and pluripotent stem cell lines.- 7.1. A historical perspective on Cre/loxP and Flp/FRT systems.- 7.2. Mechanisms of Cre/loxP and Flp/FRT recombination.- 7.3. Mutant lox and FRT sites.- 7.4. Pluripotent stem cells, an overview.- 7.5. Basic strategies for site-specific integration of transgenes via Cre/loxP and Flp/FRT systems.- 7.6 Delivery of recombinases into PSCs.- 7.7. Applications of Cre/loxP and Flp/FRT systems in the introduction of transgenes to PSCs.- 7.8. Technological improvements and future outlook.- 8 : Site-specific recombination using C31 Integrase.- 8.1 Introduction.- 8.2 C31 Integrase: Mechanism of Integration and Optimization for Heterologous Organisms.- 8.3 Use of C31 in Heterologous Organisms.- 8.4 Therapeutic Uses of C31 Integrase.- 8.5 Use of C31 in Pluripotent Stem Cells 24.- 8.6 Safety of the C31 Integrase System 28.- 8.7 The Future of C31 Integrase: Combinatorial Usage With Other Recombinases.- 9 : Modified transposases used for site-directed insertion of transgenes.- 9. 1 Introduction.- 9.2 Targeted transposition in bacteria.- 9.3 Targeted transposition in insects.- 9.4 Targeted transposition in zebrafish .- 9.5 Targeted transposition in mammalian cells.- 9.6 Conclusion-Discussion.- 10 : Targeted Plasmid Integration into the Human Genome by Engineered Recombinases.- 10.1 Introduction.- 10.2 Nonviral methods for transgene integration.- 10.3 Transgene integration by site-specific recombinases.- 10.4 Conclusions, challenges, and future directions.- Section4: Applications.- 11: Gene site-specific insertion in plants.- 11.1. Introduction.- 11.2. Approaches for the delivery of a foreign molecule into plant cells.- 11.3. Selection approaches.- 11.4. Current advances for improving targeting efficiency.- 12: Site-directed insertion of transgenes in Drosophila.- 12.1. Introduction.- 12.2. Recombinases and Integrases.- 12.3. Gene Targeting.- 12.4. Break-mediated Gene Targeting.- 12.5. Comparison of Approaches.- 13. Nuclease Mediated Targeted Genome Modification in Mammalian Cells.- 13.1. Introduction.- 13.2. Methods for Targeted Genome Engineering in Human Cells.- 13.3. Genetic Modifications Achieved with Targeted Genome Engineering.- 13.4. Non-Clinical Applications for Targeted Genome Engineering.- 13.5. Obstacles and Safety Concerns for Targeted Genome Engineering in Human Cells.- 13.6. Conclusion.- 14: Bio-applications derived from site-directed genome modification technologies.- 14.1. Introduction.- 14.2. Functional genomics.- 14.3. Drug discovery.- 14.4. Bioproduction.- 14.5. Cell transformation.- 14.6. Molecular biology and microbiology tools.- 14.7. Conclusion.