Cre-loxP based mouse models to study prionpathogenesis in the motor nervous system [Elektronische Ressource] / vorgelegt von Katja Hochgräfe
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Cre-loxP based mouse models to study prionpathogenesis in the motor nervous system [Elektronische Ressource] / vorgelegt von Katja Hochgräfe

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131 pages
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AbstandCre-loxP based mouse models to study prionpathogenesis in the motor nervous systemDissertation zur Erlangung desnaturwissenschaftlichen Doktorgradesder Bayerischen Julius-Maximilians-Universität Würzburgvorgelegt vonKatja Hochgräfeaus MelleWürzburg 2009Eingereicht am: ..........................................................Mitglieder der PromotionskommissionVorsitzender: ..........................................................Gutachter: Prof. Dr. Michael A. Klein: Prof. Dr. Erich BuchnerTag des Promotionskolloquiums: ..........................................................Doktorurkunde ausgehändigt am:....LeerseiteCONTENTSContents1 Introduction 71.1 Transmissible spongiform encephalopathies . . . . . . . . . . . . . . . . 71.2 Prion disease of humans and animals . . . . . . . . . . . . . . . . . . . 81.2.1 Animal prion diseases . . . . . . . . . . . . . . . . . . . . . . . . 81.2.2 Human prion diseases . . . . . . . . . . . . . . . . . . . . . . . 101.3 The prion concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.4 The prion protein (PrP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15C1.4.1 Primary structure and processing of PrP . . . . . . . . . . . . . 15C1.4.2 The physiological function of PrP . . . . . . . . . . . . . . . . . 16C Sc1.4.3 Structural and biochemical properties of PrP and PrP . . . . 181.4.4 Models for prion replication . . . . . . . . . . . . . . . . . . . . . 201.

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Publié le 01 janvier 2009
Nombre de lectures 8
Langue English
Poids de l'ouvrage 14 Mo

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Abstand
Cre-loxP based mouse models to study prion
pathogenesis in the motor nervous system
Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg
vorgelegt von
Katja Hochgräfe
aus Melle
Würzburg 2009Eingereicht am: ..........................................................
Mitglieder der Promotionskommission
Vorsitzender: ..........................................................
Gutachter: Prof. Dr. Michael A. Klein: Prof. Dr. Erich Buchner
Tag des Promotionskolloquiums: ..........................................................
Doktorurkunde ausgehändigt am:....LeerseiteCONTENTS
Contents
1 Introduction 7
1.1 Transmissible spongiform encephalopathies . . . . . . . . . . . . . . . . 7
1.2 Prion disease of humans and animals . . . . . . . . . . . . . . . . . . . 8
1.2.1 Animal prion diseases . . . . . . . . . . . . . . . . . . . . . . . . 8
1.2.2 Human prion diseases . . . . . . . . . . . . . . . . . . . . . . . 10
1.3 The prion concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.4 The prion protein (PrP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
C1.4.1 Primary structure and processing of PrP . . . . . . . . . . . . . 15
C1.4.2 The physiological function of PrP . . . . . . . . . . . . . . . . . 16
C Sc1.4.3 Structural and biochemical properties of PrP and PrP . . . . 18
1.4.4 Models for prion replication . . . . . . . . . . . . . . . . . . . . . 20
1.5 Prion Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
1.5.1 Neuroinvasion of prions: the spread of prions from peripheral
sites to the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
1.5.2 Prion-induced neurodegeneration . . . . . . . . . . . . . . . . . 24
1.6 Dominant-negative effect of PrP polymorphisms . . . . . . . . . . . . . 25
1.7 Cre/loxP system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
1.8 Adeno associated virus vectors . . . . . . . . . . . . . . . . . . . . . . 29
2 Aim of the study 31
3 Material 32
3.1 Laboratory equipment and software . . . . . . . . . . . . . . . . . . . . 32
3.2 Buffers and chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.3 Enzymes and synthetic oligonucleotides . . . . . . . . . . . . . . . . . 38
3.4 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.5 Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.6 DNA and Protein molecular weight markers . . . . . . . . . . . . . . . . 40
3.7 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.8 Mouse strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.8.1 C57BL/6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
0=03.8.2 Prnp (Zürich I knockout) . . . . . . . . . . . . . . . . . . . . . . 40
3.8.3 Prnp lox2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.8.4 Tga20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
0=03.8.5 Tg(SHaPrP) (Tg(SHaPrP)3922-Prnp ) . . . . . . . . . . . . . . 42
3.8.6 NF-L-Cre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
tm4(Cre)Tmj3.8.7 Hb9-Cre (B6.129S1-Mnx1 /J) . . . . . . . . . . . . . . 42
3.9 Scrapie inoculum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4CONTENTS
4 Methods 43
4.1 Animal methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Q167R4.1.1 Generation of Tg floxed LacZ-PrP mice . . . . . . . . . . . 43
Q167R4.1.2 Mating of Tg floxed LacZ-PrP mice to Hb9-Cre and NF-L-
Cre strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.1.3 Mating of Lox2 mice to Hb9-Cre and NF-L-Cre strains . . . . . . 44
4.1.4 Tail biopsy and earmarks . . . . . . . . . . . . . . . . . . . . . . 44
4.1.5 Intramuscular application of AAV-Cre vectors . . . . . . . . . . . 44
4.1.6 Scrapie inoculation of mice . . . . . . . . . . . . . . . . . . . . . 44
4.1.7 Bioassay to determine prion infectivity . . . . . . . . . . . . . . . 45
4.1.8 Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.2 Molecular biology techniques . . . . . . . . . . . . . . . . . . . . . . . . 45
4.2.1 Isolation of genomic DNA from tissue samples . . . . . . . . . . 45
4.2.2 Determination of the nucleic acid concentration . . . . . . . . . . 46
4.2.3 Polymerase chain reaction (PCR) . . . . . . . . . . . . . . . . . . 46
Q167R4.2.4 Copy number studies in Tg floxed LacZ-PrP mice . . . . . . . 48
4.2.5 Restriction enzyme digestion of DNA . . . . . . . . . . . . . . . . 50
4.2.6 Agarose gel electrophoresis of DNA . . . . . . . . . . . . . . . . 50
4.2.7 Production of adeno-associated virus vectors (dsAAV2-Cre) ex-
pressing Cre-recombinase . . . . . . . . . . . . . . . . . . . . . 51
4.3 Cell culture techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.3.1 Culture conditions, passaging and seeding of cells . . . . . . . . 52
4.3.2 Transient calcium-phosphate transfection of 293T cells . . . . . . 52
4.3.3 Fuorescence-activated cell sorting (FACS) . . . . . . . . . . . . 52
4.3.4 X-Gal assay to detect-galactosidase in 293T cells . . . . . . . 53
4.3.5 293T cell lysis for western blot analysis . . . . . . . . . . . . . . 53
4.4 Biochemical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.4.1 Preparation of tissue homogenates . . . . . . . . . . . . . . . . 54
4.4.2 Bradford assay to determine of protein concentrations . . . . . . 54
4.4.3 Proteinase-K digestion . . . . . . . . . . . . . . . . . . . . . . . . 54
4.4.4 Peptide-N-glycosidase F (PNGase F) digestion . . . . . . . . . 54
Sc4.4.5 Sodium phosphotungstic acid (NaPTA) precipitation of PrP . . 55
4.4.6 SDS polyacrylamid gel electrophoresis . . . . . . . . . . . . . . 55
4.4.7 Western blotting and immunodetection of proteins . . . . . . . . 55
4.4.8 Stripping and re-exposure of western blot membranes . . . . . . 56
4.4.9 Densitometric analysis of protein signals . . . . . . . . . . . . . . 56
4.5 Histological techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.5.1 Tissue preparation for histological analysis . . . . . . . . . . . . 57
4.5.2 X-Gal assay to detect-galactosidase (-gal) . . . . . . . . . . . 57
5CONTENTS
4.5.3 Immunohistochemistry (IHC) . . . . . . . . . . . . . . . . . . . . 58
5 Results 60
Q167R5.1 Transgenic mice with conditional expression of PrP . . . . . . . . . 60
Q167R5.1.1 Generation and characterization of Tg floxed LacZ-PrP mice 60
5.1.2 Survival and pathology of transgenic mice upon prion infection . 77
Q167R5.2 Transfer of dsAAV2-Cre into Tg floxed LacZ-PrP mice . . . . . . . . 84
5.3 Prion accumulation in spleen of neuronal PrP deficient mice . . . . . . 88
5.3.1 Characterization of neuronal PrP deficient mice . . . . . . . . . . 88
Sc5.3.2 Accumulation of PrP in spleen and spinal cord of neuronal PrP
deficient mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
5.3.3 Determination of prion infectivity titers in spleen of neuronal PrP
deficient mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
6 Discussion 93
Q167R6.1 Transgenic mice with conditional expression of PrP . . . . . . . . . 93
Q167R6.2 Transfer of dsAAV2-Cre into Tg floxed LacZ-PrP mice . . . . . . . . 99
6.3 Prion accumulation in spleen of neuronal PrP deficient mice . . . . . . 101
7 Summary 105
8 Zusammenfassung 107
References 109
9 Supplement 125
9.1 Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
9.2 Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
9.3 Resume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
9.4 Publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
9.5 Erklärung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
9.6 Danksagung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
61 INTRODUCTION
1 Introduction
1.1 Transmissible spongiform encephalopathies
Transmissible spongiform encephalopathies or prion diseases are neurodegenerative
disorders of the central nervous system (CNS) leading to motor dysfunctions, dementia
and death. Among animals the most prominent prion diseases are scrapie in sheep
and bovine spongiform encephalopathy (BSE) in cattle. Human prion diseases in-
clude Creutzfeldt-Jakob disease (CJD), Kuru, Gerstmann-Sträussler-Scheinker syn-
drome (GSS) and fatal familiar insomnia (FFI). Recently, a new variant of CJD (vCJD)
attracted the interest of the public as it was ascribed to the consumption of BSE-
contaminated products [Will et al., 1996] and to date has claimed 164 victims until
rd 1the 3 August 2009 .
Prion diseases show up with long incubation times and so far no therapy is available.
Moreover a progressive course of the disease is observed, including a slow destruc-
tion of the brain, which is accompanied by alterations in behavior, motor impairments
and cognitive decline. Beneath the characteristic neuropathological changes, such as
spongiform degeneration of the CNS, astrocytic gliosis and neuronal death, the accu-
mulation of amyloid deposits is found frequently but not necessarily in the brains o

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