Molecular genetic and phenotypic analysis of ENU-induced mutant mouse models for biomedical research [Elektronische Ressource] / Sudhir Kumar. Betreuer: Bernhard Aigner

Sudhir Kumar

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Aus dem Department für Veterinärwissenschaften
der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München


Arbeit angefertigt unter der Leitung von
Univ.-Prof. Dr. Bernhard Aigner

Molecular genetic and phenotypic analysis of
ENU-induced mutant mouse models for biomedical
research

Inaugural-Dissertation
zur Erlangung der tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität
München


von
Sudhir Kumar
aus
Majra, India

München 2011 From the Department of Veterinary Sciences
Faculty of Veterinary Medicine
Ludwig-Maximilians-University Munich


Under the supervision of Prof. Dr. Bernhard Aigner

Molecular genetic and phenotypic analysis of
ENU-induced mutant mouse models for biomedical
research

Inaugural-Dissertation
to achieve the title Doctor of Veterinary Medicine
at the Faculty of Veterinary Medicine of the
Ludwig-Maximilians-University Munich


By
Sudhir Kumar
from
Majra, India

Munich 2011
Gedruckt mit Genehmigung der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München









Dekan: Univ.-Prof. Dr. J. Braun
Berichterstatter: Univ.-Prof. Dr. B. Aigner
Korreferent: Priv.-Doz. Dr. M. Schneider









Tag der Promotion: 30.07.2011








To my beloved parents


Table of contents V
TABLE OF CONTENTS
1 Introduction 1
2 Review of the literature 2
2.1 Mice in biomedical research 2
2.2 Single gene vs. multifactorial genetic disorders 2
2.2.1 Genetic mapping of monogenic diseases 3
2.2.2 Genome-wide association studies (GWAS) 4
2.2.3 Exome sequencing 6
2.3 Mouse models for functional genome analysis 7
2.4 ENU mutagenesis 7
2.4.1 History and mechanism of action 7
2.4.2 ENU mouse mutagenesis 9
2.4.3 Spectrum of ENU-induced mutations 11
2.4.4 Outcome of the ENU mouse mutagenesis projects 12
2.5 The phenotype-driven Munich ENU mouse mutagenesis project 13
2.5.1 The clinical chemical screen for dominant and recessive mutations 13
2.5.2 Establishment of mutant lines in the clinical chemical screen 14
2.5.3 Analysis of the causative mutation 15
3 Research methodology 18
3.1 ENU-induced mutant lines analyzed in this study 18
3.1.1 Line HST014 18
3.1.2 Line HST011
3.1.3 HST015 19
3.1.4 Line CLP001
3.2 Animal husbandry and maintenance of the mutant lines 20 Table of contents VI
3.3 Analysis of the causative mutation 20
3.3.1 Line HST014 20
3.3.1.1 Linkage analysis 20
3.3.1.2 Fine mapping and selection of candidate genes 21
3.3.1.3 Analysis of the candidate genes 22
3.3.1.4 Genotyping of the animals of line HST014 23
3.3.2 Line HST011 24
3.3.2.1 Fine mapping of chromosome 1 24
3.3.2.2 Selection and analysis of the candidate gene 25
3.3.2.3 Genotyping of the animals of line HST011 26
3.3.3 Line HST015 26
3.3.3.1 Linkage analysis 26
3.3.3.2 Fine mapping of chromosome 7 27
3.3.3.3 Selection and analysis of the candidate genes 27
3.3.4 Line CLP001 28
3.3.4.1 Selection and analysis of the candidate gene 28
3.3.4.2 Genotyping of the animals of line CLP001 29
3.4 Molecular genetic methodologies 30
3.4.1 Genomic DNA isolation and analysis 30
3.4.2 RNA isolation and analysis 31
3.4.3 First strand cDNA synthesis 32
3.4.4 PCR 32
3.4.5 Elution of PCR products from the agarose gel 33
3.4.6 Sequencing of purified PCR products 33
Table of contents VII
3.5 Phenotype analysis 34
3.5.1 Blood plasma 34
3.5.2 Metabolic cage analysis
3.5.3 Morphological studies 35
3.5.4 SDS-PAGE analysis for the detection of albuminuria 35
3.5.5 Generation of a congenic line 36
3.6 Data presentation and statistical analysis of the data 36
4 Results 37
4.1 Line HST014 37
4.1.1 Linkage analysis of the causative mutation 37
4.1.2 Identification of the causative mutation 40
I27N4.1.3 Allelic differentiation of the Kctd1 mutation by PCR-RFLP 42
I27N4.1.4 Analysis of Kctd1 homozygous mutant mice 42
I27N4.1.5 Clinical chemical analysis of Kctd1 heterozygous mutant mice 43
I27N4.1.6 Urine analysis of Kctd1 heterozygous mutant mice 45
I27N4.1.7 Morphological analysis of Kctd1 heterozygous mutant mice 47
4.2 Line HST011 48
4.2.1 Re-analysis of line HST011 showed erroneous linkage analysis 48
4.2.2 Re-mapping of the causative mutation to chromosome 1 50
4.2.3 Sequence analysis of the gene Pou3f3 51
L423P4.2.4 Allelic differentiation of the Pou3f3 mutation by PCR-RFLP 52
L423P4.2.5 Clinical chemical analysis of Pou3f3 homozygous mutant mice 53
L423P4.2.6 Urine analysis of Pou3f3 homozygous mutant mice 55
L423P4.2.7 Morphological analysis of Pou3f3 homozygous mutant mice 56
Table of contents VIII
4.3 Line HST015 59
4.3.1 Linkage analysis of the causative mutation 59
4.3.2 Fine mapping of chromosome 7 60
4.3.3 Candidate genes analysis 60
4.3.4 Clinical chemical analysis of phenotypically heterozygous mutant mice 62
4.3.5 Phenotypical analysis of backcross mice 62
4.4 Line CLP001 63
4.4.1 Sequence analysis of the gene Gsdma3 63
I359N4.4.2 Allelic differentiation of the Gsdma3 mutation by ARMS-PCR 64
I359N4.4.3 Analysis of alopecia in Gsdma3 mutant mice 65
I359N4.4.4 Clinical chemical analysis of Gsdma3 mutant mice 66
I359N4.4.5 Morphological analysis of Gsdma3 mutant mice 66
4.5 Generation of congenic lines 69
5 Discusion 70
I27N5.1 Line HST014 exhibiting the mutation Kctd1 70
L423P5.2 Line HST011 exhibiting the mutation Pou3f3 72
5.3 Line HST015 established by increased plasma urea levels 74
I359N5.4 Line CLP001 exhibiting the mutation Gsdma3 74
6 Summary 77
7 Zusamenfasung 79
8 References 81
9 Acknowledgement 91
List of abbreviations IX
LIST OF ABBREVIATIONS
Aqp4 Aquaporin 4
Aqp11 Aquaporin 11
bp Base pair
cDNA Complementary deoxyribonucleic acid
Chromodomain helicase DNA binding Chd2
protein 2
DNA Deoxyribonucleic acid
dNTP Deoxyribonucleotide Triphosphate
DTT Dithiothreitol
EDTA Ethylene diamine tetraacetic acid
ENU N-ethyl-N-nitrosourea
ES Embryonic stem
Gsdma3 Gasdermin 3
h Hour
Het Heterozygous mutant
Hom Homozygous mu
Kctd1 Potassium channel tetramerization
domain-containing 1
Mb Megabase
Mep1b Meprin 1 beta
nt Nucleotide
PAGE Polyacrylamide gel electrophoresis
PCR Polymerase chain reaction
Pou3f3 POU domain, class 3, transcription
factor 3
RE Restriction endonuclease
RFLP Restriction fragment length
polymorphism List of abbreviations X
RNA Ribonucleic acid
SDS Sodium dodecyl sulphate
Sec Second
SNP Single nucleotide polymorphism
TE buffer Tris EDTA buffer
Tomt Transmembrane O-methyltransferase
Tris Tris(hydroxymethyl)aminomethane
Uromodulin Umod
Wnt11 Wingless-related MMTV integration site
11
Wt Wild-type