Genetic targeting of Cre recombinase to the murine ACTH receptor locus [Elektronische Ressource] / vorgelegt von Florian Riese

Florian Riese

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 46
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Aus dem

Max-Planck-Institut für Psychiatrie

Direktor: Prof. Dr. rer. nat. Dr. med. Florian Holsboer







Genetic Targeting of Cre Recombinase to the Murine ACTH Receptor Locus





Dissertation
zum Erwerb des Doktorgrades der Medizin
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München





Vorgelegt von

Florian Riese

aus

Hannover

Jahr

2009












Mit Genehmigung der Medizinischen Fakultät
der Universität München







Berichterstatter: Prof. Dr. rer. nat. Dr. med. Florian Holsboer


Mitberichterstatter: Prof. Dr. Rainer Rupprecht
Priv. Doz. Dr. Christoph Auernhammer


Mitbetreuung durch den
promovierten Mitarbeiter: Dr. rer. nat. Jan Deussing

Dekan: Prof. Dr. med. Dr. h. c. M. Reiser, FACR, FRCR


Tag der mündlichen Prüfung: 19.03.2009 Contents

CONTENTS

1 INTRODUCTION 5

1.1 The Hypothalamic-Pituitary-Adrenocortical Axis 5
1.1.1 Overview of Function 5
1.1.2 The HPA Axis and Depression 7

1.2 Genetic Mouse Models in Psychiatric Research 9

1.3 Manipulating the Mouse Genome 12
1.3.1 The Phenotype-Based Approach
1.3.2 Transgenic Mice 13
1.3.3 Gene Targeting 14
1.3.4 Conditional Control of Gene Expression 17

1.4 The Cre/Lox System 18
1.4.1 Overview of Function 18
1.4.2 Applications in Mouse 19
1.4.3 Cell-Type Specific Cre Expression 20
1.4.4 Inducible Cre Expression 21
1.4.5 Pitfalls of the Cre/Lox System 23

1.5 Applying the Cre/Lox System to the Adrenal Cortex 24
1.5.1 The Receptor for Adrenocorticotropic Hormone 25
1.5.2 Properties of the ACHTR Promoter 27

1.6 Aim of the Thesis 30


2 MATERIALS 31

2.1 Buffers and Solutions 31
2.1.1 Electrophoresis Buffers 31
2.1.1.1 Buffers for DNA Electrophoresis 31
2.1.1.2 RNA
2.1.2 Buffers for Southern Blotting 32
2.1.3 Buffers and Media for Bacterial and Cell Culture 33

2.2 Cell Lines 34

2.3 Oligonucleotide Sequences 35


1Contents

3 METHODS 37

3.1 Molecular Cloning Techniques 37
3.1.1 Transformation of Plasmid DNA 37
3.1.2 Isolation of Nucleic Acids 38
3.1.2.1 of Vector 38
3.1.2.2 Isolation of Genomic DNA
3.1.2.3 Isolation of Total RNA 39
3.1.3 Purification of DNA 39
3.1.3.1 Phenol/Chloroform Extraction 39
3.1.3.2 Ethanol Precipitation 40
3.1.3.3 PCR Purification 40
3.1.4 Restriction Digestion of DNA
3.1.5 Isolation of DNA Fragments 41
3.1.6 Ligation of DNA
3.1.7 Recombineering by Red/ET-Cloning 42
3.1.8 Polymerase Chain Reaction 43
3.1.8.1 Standard PCR 43
3.1.8.2 PCR Amplification of Long DNA Fragments 44
3.1.8.3 Nested 44
3.1.8.4 Multiplex PCR 45
3.1.8.5 Megaprime
3.1.8.6 Colony 45
3.1.8.7 Reverse Transcription PCR 45
3.1.8.8 Primer Design 46
3.1.9 Agarose Gel Electrophoresis 46
3.1.10 Determination of DNA/RNA Concentration 46

3.2 Blotting Techniques 47
3.2.1 Southern Blotting of Agarose Gels 47
3.2.2 Colony Hybridization 48

3.3 Cell Culture Techniques 48
3.3.1 Manipulation of Embryonic Stem Cells 48
3.3.1.1 Culture of Embryonic Mouse Fibroblast Feeder Cells 49
3.3.1.2 Culture of Embryonic Stem Stem Cells 50
3.3.1.3 Electroporation of Embryonic Stem Cells 50
3.3.1.4 Identification of Homologously Recombined ES Cells 51
3.3.1.5 Preparation of ES Cells for Blastocyst Injection 51
3.3.2 Culture of Y1 Adrenocortical Cells 52








2Contents

4 RESULTS 53

4.1 Generation of Constructs pPNTflpCremyctagPml and
pPNTflpCreERT2Pml 53
4.1.1 Modification of the Universal Gene Targeting Vector pPNTflp 53
4.1.2 Cloning of Homologous Arms 56
4.1.2.1 Generation of the 5’ Homologous Arm 56
4.1.2.2 Fusion of 5’ HA to Cre Recombinases 56
4.1.2.3 Generation of the 3’ 57
4.1.3 Insertion of the 3’ HA into the Targeting Vector pPNTflpfseSgr 58
4.1.4 Completion of Constructs pPNTflpCremyctagPml and
pPNTflpCreERT2Pml 59

4.2 Embryonic Stem Cell Culture 60

4.3 Screening of ES Cell Clones for Construct Integration 61

4.4 Generation of ACTHR-CE2 Mice 62

4.5 Establishment of a Genotyping PCR for ACTHR-CE2 Mice 63

4.6 Characterization of the Targeted ACTHR Locus 64

4.7 ation of ACTHR-CE2 Mice 66
4.7.1 Evaluation for Flp Mediated Selection Cassette Excision 66
4.7.2 Phenotyping of ACTHR-CE2 Mice 66
4.7.3 Evaluation of Cre Expression by RT-PCR and Western Blotting 66

4.8 Generation of Constructs pMC2RcreMYC and pMC2RcreERT2 67
4.8.1 Generation of Homologous Sequences and Cre 68
4.8.1.1 5’ Homologous Sequence and Cre Open Reading Frame 68
4.8.1.2 3’ Homologous Sequence 68
4.8.2 Generation of Cloning Cassettes 69
4.8.2.1 Improved Yellow Fluorescent Protein (Fragment 1) 69
4.8.2.2 SV40 Polyadenylation Signal (Fragment 2) 70
4.8.2.3 Neomycin Resistance (Fragment 3) 70
4.8.2.4 Ampicillin Resistance 4)
4.8.2.5 Mutated Estrogen Receptor LBD (ERT2) 70
4.8.3 Assembly of Vectors pMC2RcreMYC and pMC2RcreERT2 71
4.8.3.1 Ligation of Fragments 1 and 2 into p5’HAPCR 71
4.8.3.2 Ligation of CreERT2 C-Terminus and ERT2
into p5’HAF12 72
4.8.3.3 Ligation of CreMYC C-Terminus into p5’HAF1F2 73
4.8.3.4 Ligation of Fragments 3 and 4 into p3’HAPCR 74
4.8.3.5 Completion of the Targeting Constructs pMC2RcreMYC and
pMC2RcreERT2 4

4.9 Recombineering of pMC2RcreMYC and pMC2RcreERT2 into the Targeting
Cosmid 76
3Contents

5 DISCUSSION 78

5.1 Overview 78

5.2 Rationale

5.3 Driving Cre Expression by the ACTHR Promoter 80

5.4 Strategy One: Classical Knock-In Vectors 83
5.4.1 Choice of Constituitively Active Cre Variant 84
5.4.2 Choice of Inducible Cre Variant 84
5.4.3 Selection Cassettes 86
5.4.4 ACTHR-CE2 Mice 87

5.5 Strategy Two : Recombineering Vectors 92
5.5.1 Cosmid Recombineering 93
5.5.2 Fluorescent Marker 96

5.6 Outlook 97
5.6.1 Towards the Generation of ACTHR Cre Cosmid Mice 97
5.6.2 Targets for ACTHR Cre Mice 99
5.6.2.1 CRHR1 as Regulator of Glucocorticoid Secretion 100
5.6.2.2 A Mouse Model for Adrenocortical Carcinoma 102

5.7 Conclusion 103

6 SUMMARY 104

7 ZUSAMMENFASSUNG 106

8 REFERENCES 109

9 ACKNOWLEDGMENTS 128

10 CURRICULUM VITAE 129
4Introduction

1 Introduction
1.1 The Hypothalamic-Pituitary-Adrenocortical Axis
1.1.1 Overview of Function
All organisms strive towards maintaining their homeostasis, i.e. the dynamic
equilibrium of their internal milieus that is essential for survival. The challenge of
homeostasis by internal or external factors is classically referred to as “stress”.
“Stress reaction” is the response of an organism to stress and spans from local
biochemistry to global behavior. In mice and men, the sympathetic nervous system
and the hypothalamic-pituitary-adrenocortical (HPA) system are the central
regulators of the stress response (de Kloet et al., 2005). The HPA axis is formed by
three main structures: the hypothalamus, the pituitary and the adrenal gland.

The hypothalamus is located below the thalamus along the walls of the third ventricle
and has multiple roles in the maintenance of homeostasis. For the mediation of the
stress response, parvocellular neurons from the paraventricular part of the
hypothalamus are of crucial importance. They secrete corticotropin-releasing
hormone (CRH, also CRF: corticotropin-releasing factor) into the pituitary portal
vessels. The pituitary gland is situated in the sella turcica and consists of three lobes,
an anterior, an intermediate and a posterior lobe. Upon CRH stimulation, endocrine
cells from the anterior lobe of the pituitary release adrenocorticotropic hormone
(ACTH) into the general circulation. With the blood flow, ACTH reaches the adrenal
glands which are located on the upper pole of the kidneys. The adrenal glands
comprise two organ compartments, the adrenal medulla and the adrenal cortex.
While the medulla forms part of the sympathetic nervous system, the cortex is the
body’s major source of steroid hormones. Of these, mineralocorticoids are
synthezised in the glomerular layer, glucocorticoids in the fasciculate lay