Phenotypic variability in monogenic disorders involving skeletal malformations [Elektronische Ressource] / vorgelegt von Wibke Schwarzer

Wibke Vonbonin

Sujets

• Biologie

Informations

• Publié par: freie_universitat_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 39
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

Phenotypic variability in monogenic disorders in-
volving skeletal malformations




Dissertation zur Erlangung des akademischen Grades des
Doktors der Naturwissenschaften (Dr. rer. nat.)



eingereicht im Fachbereich Biologie, Chemie, Pharmazie
der Freien Universität Berlin



vorgelegt von
Wibke Schwarzer
aus Baden%Baden




Juni 2010
Diese Dissertation wurde in der Zeit vom 15.05.2006 bis zum 30.04.2010 in der Ar%
beitsgruppe von Prof. Dr. Stefan Mundlos am MPI für Molekulare Genetik in Berlin
angefertigt.

1. Gutachter: Prof. Dr. Stefan Mundlos
Max%Planck%Institut für Molekulare Genetik
Ihnestraße 73, 14195 Berlin
Tel.: [phone]
Email: [e-mail]

2. Gutachter: Prof. Dr. Wolfgang Schuster
Institut für Biologie
Freie Universität Berlin
Albrecht%Thaer%Weg 6 , 14195 Berlin
Tel.: [phone]
Email: [e-mail]%berlin.de











Disputation am: 28.09.2010





“It is owing to wonder that people began to philosophize, and wonder
remains the beginning of knowledge.”
Aristoteles







Selbstständigkeitserklärung
Hiermit erkläre ich, dass ich die vorliegende Arbeit selbst angefertigt und keine an%
deren, als die hier angegebenen Hilfsmittel verwendet habe. Ich versichere, dass ich
diese Arbeit weder in dieser noch in einer anderen Form bei einer anderen Prüfungs%
behörde eingereicht habe.

Berlin, den 03. Juni 2010



%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
Wibke Schwarzer

Table of Content
1 Introduction ............................................................................................................ 1
1.1 ROR2 % the genetic cause for two developmental disordesr ............................. 2
1.1.1 Receptor tyrosine kinase%like orphan receptor 2 (ROR2) ..................... 2
1.1.2 Mutations in ROR2 cause either Recessive Robinow Syndrome (RRS)
or dominant Brachydactyly Type B1 (BDB1) ...................................... 3
1.1.3 Ror2 mouse models for RRS and BDB1 ............................................... 7
1.2 Split hand and foot malformation (SHFM) ....................................................... 8
1.2.1 SHFM is a clinically variable and genetically heterogeneous
disorder.. ................................................................................................ 8
1.2.2 Molecular pathogenesis of ectrodactylies ........................................... 10
1.2.3 The dactylaplasia mouse % a model for human SHFM3 ..................... 13
1.3 Thesis objectives ............................................................................................. 18
2 Material ................................................................................................................. 19
2.1 Instruments ...................................................................................................... 19
2.2 Chemicals ........................................................................................................ 20
2.3 Buffers ............................................................................................................. 20
2.4 Kits ............................................................................................................. 20
2.5 Enzymes .......................................................................................................... 21
2.6 Bacterial strains ............................................................................................... 21
2.7 Expression constructs and vectors .................................................................. 21
2.8 Primer ............................................................................................................. 21
2.8.1 ROR2 mutagenesis primer .................................................................. 21
2.8.2 Primer for qPCR .................................................................................. 22
2.8.3 Primer for probe amplification ............................................................ 22
2.8.4 Primer for microsatellite profiling ....................................................... 22
2.8.5 Primer for SNP profiling ..................................................................... 23
2.9 Cultured cell lines ........................................................................................... 26
2.10 Antibodies ...................................................................................................... 26
2.10.1 Primary antibodies ............................................................................... 26
2.10.2 Secondary antibodies ........................................................................... 27
2.11 Animals .......................................................................................................... 27
2.12 Software ......................................................................................................... 27
2.13 Internet resources ........................................................................................... 28
3 Methods ................................................................................................................. 29
3.1 Molecular Biological Methods ....................................................................... 29
3.1.1 DNA Isolation ..................................................................................... 29
3.1.2 RNA isolation ..................................................................................... 30
3.1.3 Generation of cDNA ........................................................................... 30
3.1.4 Linear mRNA amplification ............................................................... 30
3.1.5 Polymerase chain reaction (PCR) ....................................................... 31
3.1.6 DNA sequencing ................................................................................. 36
3.2 Cell culture ...................................................................................................... 37
3.2.1 Thawing of cells .................................................................................. 37
3.2.2 Splitting of cells .................................................................................. 37
3.2.3 Cryopreservation of cells .................................................................... 37
3.2.4 Cell number determination ................................................................. 38
3.2.5 Cell transfection .................................................................................. 38
3.2.6 Generation of stable cell lines ............................................................. 38
3.2.7 Immunocytochemistry (ICC) .............................................................. 39
3.2.8 Cell%surface protein biotinylation ..................................................... 39
3.3 Biochemical methods ...................................................................................... 40
3.3.1 Determination of protein concentration .............................................. 40
3.3.2 SDS%PAGE ....................................................................................... 40
3.3.3 Western Blot (WB) ............................................................................. 41
3.4 Animal sample preparation ............................................................................. 42
3.4.1 Embryo dissection ............................................................................... 42
3.4.2 AER dissection.................................................................................... 42
3.4.3 Skeletal preparations ........................................................................... 42
3.5 Histology ......................................................................................................... 43
3.5.1 Paraffin embedding and sectioning ..................................................... 43
3.5.2 Immunohistochemistry (IHC) ............................................................. 43
3.5.3 RNA In situ hybridisation (ISH) ......................................................... 44 3.6 Bioinformatics and data processing ................................................................ 46
3.6.1 SNP data and haplotype analysis ........................................................ 46
3.6.2 3D%reconstruction of embryonic limbs ............................................. 47
4 Results ................................................................................................................... 48
4.1 Molecular analysis of ROR2 mutations leading to dominant BDB1, recessive
RRS and intermediary phenotypes .................................................................. 48
4.1.1 A novel recessive mutation in ROR2 exhibiting features of RRS and
severe brachydactyly ........................................................................... 48
4.1.2 Exact copies of human ROR2 were generated for molecular
analysis.... ............................................................................................ 49
4.1.