An inducible RNAi system for the functional dissection of genes in the mouse [Elektronische Ressource] / by Joana Alves Vidigal
158 pages
English

An inducible RNAi system for the functional dissection of genes in the mouse [Elektronische Ressource] / by Joana Alves Vidigal

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158 pages
English
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An Inducible RNAi system for the functional dissection of genes in the mouse Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of the Freie Universität Berlin by Joana Alves Vidigal from Lisbon, Portugal December 2010 The work here described was performed at the Max-Planck Institute for Molecular Genetics in Berlin between February 2007 and December 2010 under the supervision of Prof. Dr. Bernhard G. Herrmann and Dr. Markus Morkel st1 Reviewer: Prof. Dr. Bernhard G. Herrmann Max-Planck Institute for Molecular Genetics Ihnestrasse 73 D-14195 Berlin nd2 Reviewer: Prof. Dr. Stephan Sigrist Institute of Biology - Freie Universität Berlin Takustr. 6, 14195 Berlin Date of defence: 11/02/2011 Aos meus pais e aos meus irmãos Table of Contents 1  INTRODUCTION 11 1.1  An introduction to the mouse as a model system 12 1.2  The molecular revolution of mouse genetics 13 1.3  The “knockout mouse” 14 1.3.1  Isolation of Embryonic Stem Cells 14 1.3.2  Genetic engineering by homologous recombination. 15 1.4  Further developments in transgenic techniques 18 1.4.1  Site-specific recombination systems 18 1.4.2  Inducible expression systems 19 1.4.3  RNAi: an alternative to traditional gene targeting 22 1.5  RNA interference and its triggers 24 1.

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Publié par
Publié le 01 janvier 2010
Nombre de lectures 22
Langue English
Poids de l'ouvrage 15 Mo

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An Inducible RNAi system for the
functional dissection of genes in the
mouse

Inaugural-Dissertation
to obtain the academic degree
Doctor rerum naturalium (Dr. rer. nat.)
submitted to the Department of Biology, Chemistry and Pharmacy
of the Freie Universität Berlin






by
Joana Alves Vidigal
from Lisbon, Portugal
December 2010
The work here described was performed at the
Max-Planck Institute for Molecular Genetics in Berlin
between February 2007 and December 2010
under the supervision of Prof. Dr. Bernhard G. Herrmann and Dr. Markus Morkel

















st1 Reviewer: Prof. Dr. Bernhard G. Herrmann
Max-Planck Institute for Molecular Genetics
Ihnestrasse 73 D-14195 Berlin

nd2 Reviewer: Prof. Dr. Stephan Sigrist
Institute of Biology - Freie Universität Berlin
Takustr. 6, 14195 Berlin

Date of defence: 11/02/2011





Aos meus pais
e
aos meus irmãos


Table of Contents
1  INTRODUCTION 11 
1.1  An introduction to the mouse as a model system 12 
1.2  The molecular revolution of mouse genetics 13 
1.3  The “knockout mouse” 14 
1.3.1  Isolation of Embryonic Stem Cells 14 
1.3.2  Genetic engineering by homologous recombination. 15 
1.4  Further developments in transgenic techniques 18 
1.4.1  Site-specific recombination systems 18 
1.4.2  Inducible expression systems 19 
1.4.3  RNAi: an alternative to traditional gene targeting 22 
1.5  RNA interference and its triggers 24 
1.6  MicroRNAs 26 
1.6.1  MicroRNA structure and expression 26 
1.6.2  miRNA Maturation 27 
1.6.3  Gene silencing by miRNAs 28 
1.7  siRNAs 30 
1.7.1  Gene silencing by siRNAs 30 
1.7.2  siRNA triggers in mammals 31 
1.8  RNAi in the mouse embryo 33 
1.9  Goal 36 
2  MATERIALS AND METHODS 37 
2.1  Mouse strains and animal husbandry 38 
2.2  Generation of the RNAi vector system 39 
2.2.1  Recipient Constructs 39 
2.2.2  Exchange Vectors 39 
2.2.3  In vitro validation of shRNA-mirs 40 
2.3  Generation of Modified ES cells 41 
2.3.1  ES cell procedures 41 
2.3.2  Screening of ES cell clones by Southern blot 44 
2.4  Generation of Transgenic Mice 46 
2.4.1  Generation of full-ES cell derived embryos by tetraploid complementation 47 
2.4.2  Generation of adult chimeric mice by diploid complementation 48 
2.4.3  Animal Genotyping by PCR 48 
2.5  RNA Molecular Biology Techniques 50 
2.5.1  Northern Blot Analysis of small RNAs 50 
2.5.2  Quantitative Real Time PCR 52 
2.5.3  cDNA microarray analysis 54 
2.6  Histology 57 
2.6.1  Standard histology procedures 57 
2.6.2  Whole-mount in situ hybridization 59 2.6.3  Immunohistochemistry on sections 62 
2.6.4  Skeleton staining 63 
2.7  Primers, general chemicals and solutions 64 
2.7.1  PCR Primers 64 
2.7.2  General solutions 64 
2.7.3  General Chemicals 65 
3  RESULTS – PART I 67 
3.1  Experimental contributions 67 
3.2  An integrated vector system to target the ROSA26 locus by RMCE 68 
3.2.1  Overview of the vector system 68 
3.2.2  Initial targeting of the ROSA26 locus 69 
3.2.3  Integration of transgenes in the modified ROSA26 by RMCE 71 
3.2.4  In vitro expression of a transgene from the ROSA26 locus 72 
3.2.5  In vivo expression of transgenes from the ROSA26 locus 72 
3.2.6  Insulation of transgenes in the ROSA26 locus. 75 
3.3  Knockdown of genes from the ROSA26 locus 77 
3.3.1  Proof of principle: knockdown of brachyury 77 
3.3.2  In vitro tests of shRNA-mirs targeting brachyury 79 
3.3.3  Integration of T knockdown constructs in ROSA26S 80 
3.3.4  Expression and processing of shRNA-mirs from the ROSA26 locus 80 
3.3.5  In vivo knockdown of brachyury 82 
3.3.6  T shRNA-mir expression and processing in embryos 83 
3.3.7  System validation: in vivo knockdown of Foxa2 84 
3.3.8  System validation: in vivo knockdown of Noto 86 
3.4  Temporally controlled in vivo gene silencing 89 
3.4.1  tTS leads to irreversible transgene silencing during mouse development 89 
3.4.2  rtTA allows temporal control over knockdown 89 
3.5  Transcriptome analysis of the KD1-T RNAi model 92 
3.5.1  Deregulated genes in KD1-T caudal ends 92 
3.5.2  Knockdown occurs in the absence of off-target effects 93 
3.6  Summary and Conclusion 96 
4  RESULTS – PART II 97 
4.1  Experimental contributions 97 
4.2  KD3-T induced embryos survive mid-gestation and develop an ectopic neural tube 98 
4.3  KD3-T mutants have spina bifida 100 
4.4  KD3-T mutants display urorectal defects 101 
4.5  Summary and Conclusion 104 
5  RESULTS – PART III 106 
5.1  Experimental contributions 106 
5.2  Brachyury is required for the differentiation but not maintenance of the notochord 107 
5.3  Gross morphology of E16.5 KD4-T embryos induced at E8.5 109 
5.4  Skeletal defects in KD4-T embryos 110 5.5  Loss of Brachyury affects neural crest cell migration 113 
5.6  Analysis of chondrogenic markers in KD4-T mutants 116 
5.7  Brachyury is required for nucleus pulposus differentiation 118 
5.8  Summary and Conclusion 120 
6  DISCUSSION 122 
6.1  An inducible system to knockdown genes in the mouse 123 
6.1.1  Ubiquitous and tissue specific knockdown 124 
6.1.2  Why is the KD3-T phenotype milder than the KD2-T phenotype? 126 
6.1.3  Can knockdown be achieved in all tissues of a mouse embryo? 126 
6.1.4  Argonaute 2 and the role of small noncoding RNAs in mouse development 128 
6.2  Analysis of T knockdown models 131 
6.2.1  T and the uro-rectal-caudal syndrome 131 
6.2.2  Role of brachyury during skeletal development 132 
6.2.3  T and the induction of neural crest cells 133 
6.2.4  T and the induction of mesoderm-derived mesenchymal cells 135 
6.2.5  T in the development and disease of nuclei pulposi 137 
7  SUMMARY 140 
8  ZUSAMMENFASSUNG 141 
9  ABBREVIATIONS 142 
10  ACKNOWLEDGMENTS 144 
11  PUBLICATIONS 145 
12  CURRICULUM VITAE 146 
13  BIBLIOGRAPHY 148 

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