Comprehensive phenotyping of two mouse mutants reveals a potential novel role of G protein-coupled receptor 30 [Elektronische Ressource] / Luca Meoli. Gutachter: Patricia Ruiz ; Alf Hamann ; Ulrich Kintscher
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Comprehensive phenotyping of two mouse mutants reveals a potential novel role of G protein-coupled receptor 30 [Elektronische Ressource] / Luca Meoli. Gutachter: Patricia Ruiz ; Alf Hamann ; Ulrich Kintscher

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Max-Planck Institut für Molekulare Genetik Center for Cardiovascular Research, Charité Universitätsmedizin Berlin Comprehensive phenotyping of two mouse mutants reveals a potential novel role of G protein-coupled receptor 30 Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium im Fach Biologie eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Universität zu Berlin von Dipl.-Biologe Luca Meoli Präsident der Humboldt-Universität zu Berlin: Prof. Dr. Dr. h.c. Christoph Markschies Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I: Prof. Dr. Andreas Herrmann Gutachter/innen: 1) Prof. Patricia Ruiz 2) Prof. Alf Hamann 3) Prof. Ulrich Kintscher Tag der mündl. Prüfung: 03.08.2010 Ai miei genitori Zusammenfassung Abstract Abbreviations 1 Introduction______________________________________________________________ 1 1.1 G protein-coupled receptors __________________________________________________ 1 1.1.1 General considerations ____________________________________________________________ 1 1.1.2 G protein activation 3 1.1.3 G proteins ______________________________________________________________________ 5 1.2 GPCRs in pharmacology _____________________________________________________ 6 1.3 G protein coupled receptor 30 _________________________________________________ 7 1.3.

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Publié par
Publié le 01 janvier 2011
Nombre de lectures 32
Poids de l'ouvrage 4 Mo

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Max-Planck Institut für Molekulare Genetik
Center for Cardiovascular Research, Charité Universitätsmedizin Berlin




Comprehensive phenotyping of
two mouse mutants reveals a potential novel role
of G protein-coupled receptor 30

Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium
im Fach Biologie
eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von Dipl.-Biologe
Luca Meoli




Präsident der Humboldt-Universität zu Berlin:
Prof. Dr. Dr. h.c. Christoph Markschies
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I:
Prof. Dr. Andreas Herrmann

Gutachter/innen:
1) Prof. Patricia Ruiz
2) Prof. Alf Hamann
3) Prof. Ulrich Kintscher

Tag der mündl. Prüfung: 03.08.2010

Ai miei genitori

Zusammenfassung
Abstract
Abbreviations
1 Introduction______________________________________________________________ 1
1.1 G protein-coupled receptors __________________________________________________ 1
1.1.1 General considerations ____________________________________________________________ 1
1.1.2 G protein activation 3
1.1.3 G proteins ______________________________________________________________________ 5
1.2 GPCRs in pharmacology _____________________________________________________ 6
1.3 G protein coupled receptor 30 _________________________________________________ 7
1.3.1 Gpr30 signalling _________________________________________________________________ 7
1.3.2 Gpr30 subcellular localization and ligands_____________________________________________ 9
1.3.3 Gpr30 in vivo function ___________________________________________________________ 11
1.4 Mouse genome manipulations: transgenic and knockout mice _____________________ 17
1.4.1 Transgenic mice ________________________________________________________________ 17
1.4.2 Knockout mice 18
1.4.2.1 ES cells and gene targeting strategies____________________________________________ 18
1.4.2.2 1.4.2.2 The chimera and its offspring 20
1.4.2.3 Recombinase based approaches ________________________________________________ 21
1.5 GPCR knockout mice _______________________________________________________ 23
1.5.1 Gpr30-T181 Deltagen mice 23
1.5.2 SHG17 Artemis mice ____________________________________________________________ 25
1.6 Phenotyping strategies ______________________________________________________ 26
1.6.1 Primary screen _________________________________________________________________ 26
1.6.2 Secondary screen _______________________________________________________________ 28
1.7 Estrogen receptors in metabolic diseases and associated cardiovascular disorders _____ 29
1.8 High fat diet mouse model ___________________________________________________ 32
1.9 Assessment of endocrine and cardiovascular function in mice______________________ 34
2 Aims of the study _________________________________________________________ 37
3 Materials and Methods ____________________________________________________ 38
3.1 Gpr30 mutant mouse models _________________________________________________ 38
3.1.1 Gpr30-T181 Deltagen mice _______________________________________________________ 38
3.1.2 SHG17 Artemis mice ____________________________________________________________ 39
3.1.3 Genotyping of Gpr30-T181 Deltagen mice ___________________________________________ 39
3.1.3.1 Materials __________________________________________________________________ 39
3.1.3.2 Method ___________________________________________________________________ 41
3.1.4 Southern Blotting _______________________________________________________________ 42
3.1.4.1 Materials 42
3.1.4.2 Method 43
3.1.5 RNA extraction_________________________________________________________________ 44
3.1.5.1 Materials 44
3.1.5.2 Method 45
3.1.6 Reverse transcription ____________________________________________________________ 45
3.1.6.1 Materials 45
3.1.6.2 Method 45
3.1.7 Real Time PCR 46
3.1.7.1 Materials 46
3.1.7.2 Method ___________________________________________________________________ 46
3.1.8 SHG17 Artemis mice genotyping___________________________________________________ 47
3.1.8.1 Materials __________________________________________________________________ 47
3.1.8.2 Method ___________________________________________________________________ 47
3.2 Primary screen ____________________________________________________________ 48
3.2.1 Microarray RNA analysis_________________________________________________________ 49
3.2.1.1 Materials __________________________________________________________________ 49
3.2.1.2 Method 49
3.3 Secondary screen___________________________________________________________ 52
3.3.1 Groups _______________________________________________________________________ 52
3.3.2 Diets _________________________________________________________________________ 53
3.3.2.1 Materials 53
3.3.2.2 Method ___________________________________________________________________ 53
3.3.3 Body weight and body mass composition ____________________________________________ 54
3.3.3.1 Materials 54
3.3.3.2 Method 54
3.3.4 Intraperitoneal glucose tolerance test (IPGTT)_________________________________________ 54
3.3.4.1 Materials __________________________________________________________________ 54
3.3.4.2 Method 54
3.3.5 Echocardiography_______________________________________________________________ 56
3.3.5.1 Materials 56
3.3.5.2 Method 56
3.3.6 Electrocardiogram (ECG)_________________________________________________________ 64
3.3.6.1 Materials: _________________________________________________________________ 64
3.3.6.2 Method ___________________________________________________________________ 64
3.3.7 Blood chemistry ________________________________________________________________ 66
3.3.7.1 Materials 66
3.3.7.2 Method 66
3.3.8 Mouse housing 66
3.4 Statistical analysis __________________________________________________________ 67
3.4.1 Materials______________________________________________________________________ 67
3.4.2 Method _______________________________________________________________________ 67
4 Results _________________________________________________________________ 68
4.1 Gpr30 mutant mouse models _________________________________________________ 68
4.2 Molecular characterization of Gpr30-T181 Deltagen mice_________________________ 68
4.2.1 Genotyping ____________________________________________________________________ 68
4.2.2 Southern blotting _______________________________________________________________ 69
4.2.3 Real Time PCR 70
4.3 SHG17 Artemis mice genotyping______________________________________________ 72
4.4 Gpr30-T181 Deltagen mice phenotypic assessment _______________________________ 73
4.4.1 Primary screen 73
4.4.2 Secondary screen 80
4.4.2.1 Body weight and body mass composition_________________________________________ 81
4.4.2.2 Intraperitoneal Glucose Tolerance test (IPGTT)____________________________________ 82
4.4.2.3 Echocardiography ___________________________________________________________ 84
4.4.2.4 Blood chemistry ____________________________________________________________ 87
4.5 SHG17 Artemis mice phenotypic assessment 92
4.5.1 Thymus gene expression _________________________________________________________ 93
4.5.2 Body weight and body mass composition ____________________________________________ 94
4.5.3 Intraperitoneal glucose tolerance test ________________________________________________ 96
4.5.4 Echocardiography_______________________________________________________________ 97
5 Discussion ______________________________________________________________ 98
5.1 Gpr30 mutant mouse models _________________________________________________ 98
5.2 Primary screen 99
5.2.1 Immunological screen____________________________________________________________ 99
5.2.2 Behavioural screen _____________________________________________________________ 102
5.3 Secondary screen__________________________________________________________ 103
5.3.1 Metabolic screen_______________________________________________________________ 103
5.3.2 Cardiovascular screen___________________________________________________________ 104
5.3.3 Clinical chemistry______________________________________________________________ 105
5.4 An estrogen receptor can do more____________________________________________ 108
5.5 Conclusions and outlook____________________________________________________ 110
References 114
Table of Figures __________________________________________________________ 123
List of Tables ____________________________________________________________ 124
Acknowledgements ________________________________________________________ 125
Eidesstattliche Erklärung___________________________________________________ 126
Publications _____________________________________________________________ 127
Abstracts ________________________________________________________________ 128
Zusammenfassung

In den späten neunziger Jahren wurde der G Protein-gekoppelte Rezeptor 30 (Gpr30) von

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