Hypothalamic gene expression profiling in mouse strains susceptible or resistant to diet-induced obesity [Elektronische Ressource] / vorgelegt von Lianxing Yang

Yang Ying

Sujets

• Gesundheit

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2005
• Nombre de lectures: 25
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Hypothalamic gene expression profiling in mouse
strains susceptible or resistant to diet-induced obesity



Dissertation

zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)



dem
Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von



Lianxing Yang
aus Hebei, V. R. China


Marburg/Lahn, 2004











Vom Fachbereich Biologie .
der Philipps-Universität Marburg als Dissertation am 31. 12. 2004 angenommen.

Erstgutachter HD Dr. Martin Klingenspor .
Zweitgutachter Prof. Dr. Renate Renkawitz-Pohl .
Tag der mündlichen Prüfung am 13. 01. 2005 .










Contents

Zusammenfassung .........................................................................................................................1
1 Summary...............................................................................................................................3
2 Introduction...........................................................................................................................5
2.1 Epidemiology of obesity................................................................................................5
2.2 Effects of obesity...........................................................................................................6
2.3 Etiology.........................................................................................................................7
2.3.1 External factors......................................................................................................7
2.3.2 Internal .......................................................................................................8
2.4 Hypothalamus................................................................................................................9
2.5 Animal model for research ..........................................................................................12
2.6 Aim of this study13
3 Materials and Methods ........................................................................................................14
3.1 Diet experiment...........................................................................................................14
3.2 RNA manipulations.....................................................................................................15
3.2.1 RNA isolation
3.2.2 RNA electrophoresis...........................................................................................15
3.2.3 RNA transfer16
3.2.4 Northern hybridization........................................................................................
3.3 DNA manipulations17
3.3.1 Genomic DNA isolation......................................................................................
3.3.2 Plasmid DNA preparation from E. coli cells.......................................................17
3.3.3 Precipitation of plasmid DNA.............................................................................18
3.3.4 DNA electrophoresis18
3.3.5 Digestion of DNA by restriction endonucleases .................................................19
3.3.6 DNA isolation from agarose gel..........................................................................19
3.3.7 PCR.....................................................................................................................19
3.3.8 PCR purification..................................................................................................21
3.3.9 DNA ligation.......................................................................................................
3.3.10 Transformation of E. coli ....................................................................................21
3.3.11 DNA sequencing.................................................................................................22
3.4 RZPD filter hybridization............................................................................................
3.4.1 Quality control of filter........................................................................................22
3.4.2 Complex hybridization23
3.4.2.1 Preparation of complex cDNA samples ..........................................................24
3.4.2.2 Pre-hybridization.............................................................................................25
3.4.2.3 Complex hybridization....................................................................................26
3.4.2.4 Post-hybridization...........................................................................................26
3.5 Affymetrix GeneChip hybridization............................................................................26
3.5.1 RNA isolation......................................................................................................27
3.5.2 cDNA synthesis...................................................................................................
3.5.2.1 First-strand cDNA synthesis............................................................................27
3.5.2.2 Second-strand cDNA synthesis .......................................................................27
3.5.2.3 Cleanup of double-strand cDNA.....................................................................28
3.5.3 cRNA synthesis28
3.5.4 Cleanup and quantification of biotin-labeled cRNA...........................................
3.5.5 Fragmenting the cRNA for target preparation.....................................................29
3.5.6 GeneChip hybridization.......................................................................................29
iii Contents

3.5.7 Post-hybridization...............................................................................................30
3.6 In Situ hybridization....................................................................................................31
3.6.1 Brain sectioning...................................................................................................31
3.6.2 Glass slide preparation – silanization..................................................................
3.6.3 Preparation of the probe ......................................................................................31
3.6.3.1 Linearization of DNA template from section 3.3.3.........................................31
3.6.3.2 In Vitro transcription (IVT).............................................................................32
3.6.4 Pre-hybridization.................................................................................................33
3.6.5 Hybridization.......................................................................................................
3.6.6 Post-hybridization34
3.6.7 Signal detection
3.7 Quantitative real-time RT-PCR...................................................................................35
3.7.1 First-strand cDNA synthesis................................................................................35
3.7.2 Primer design
3.7.3 Real-time RT-PCR protocol and program...........................................................36
3.8 Data analysis................................................................................................................37
3.8.1 Data analysis I .....................................................................................................37
3.8.2 Data analysis II....................................................................................................38
3.8.3 Data analysis III...................................................................................................39
3.9 Post analysis..39
3.10 Single nucleotide polymorphism (SNP) analysis........................................................40
4 Results.................................................................................................................................41
4.1 Diet induced obesity in mice .......................................................................................41
4.1.1 Body mass...........................................................................................................41
4.1.2 Energy intake.......................................................................................................43
4.1.3 Body fat...............................................................................................................44
4.1.4 Tissue mass..........................................................................................................46
4.1.5 Litter size.............................................................................................................48
4.2 Gene expression study.................................................................................................50
4.2.1 Data analysis