Strategies to identify and further characterize novel and known genes involved in regulation of skeletogenesis [Elektronische Ressource] / Silke Schlaubitz

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2007
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

‘Strategies to identify and
further characterize novel
and known genes
involved in regulation of
skeletogenesis’




Ph.D. Thesis
to accomplish

Doctorate Degree in Science

at the Faculty of Biology

Johannes Gutenberg-University of Mainz
in Mainz , Germany


Silke Schlaubitz



born in Düsseldorf, Germany


Mainz, Germany, 2007




































Dean:
1. Correspondent:
2. Correspondent:
thOral examination: December 04 , 2007 _______________________________________________________Table of Contents
Index of figures
Index of tables


1. Introduction.........................................................................................….. 1
1.1.Endochondral and intramembranous bone formation.............… 2
1.2. Patterning during vertebrate limb bud development…………… 8
1.2.1. D/V patterning of the apical ectodermal ridge
(AER) and the progressive zone model......................……… 12
1.3. Lmx1b....................................................................................… 14
1.3.1. Discovery of Lmx1b................................… 14
1.3.2. Lmx1b expression and knockout model...................… 15
1.3.3. Downstream targets of Lmx1b.................................… 17
1.3.4. LMX1B and Nail-Patella-Syndrome..........................… 18
1.4. Genitopatellar Syndrome........................................… 19
1.5. Specific Aims..........................................................................… 20

2. Methods............................................................….................................... 22
2.1. DNA Isolation........................................................................…. 22
2.1.1. Isolation of genomic DNA from blood.......................… 22
2.1.2. Isolation of plasmid DNA……………………………….. 22
2.1.3. Isolation of genomic DNA from mouse
tissues for genotyping PCR....……........................................ 22
2.2. RNA Isolation.........................................................................… 23
2.2.1. Isolation of total RNA from tissues or cells...............… 23
2.2.2. mRNA isolation from total RNA....................24
2.2.3. RNA preparation from prenatal limb
buds for microarray experiments..............……...................... 24
2.2.4. RNA preparation from differentiated and
dedifferentiated chrondrocytes...........................................… 24
2.2.5. Laser Capture Microscopy and RNA preparation
from captured cells................................................................. 25
2.3. DNA and RNA standard methods.............................… 26
2.3.1. Determination of DNA, cDNA and RNA
concentrations....................................................................… 26
2.3.2. Agarose gel electrophoresis of DNA and RNA.........… 26
2.3.3. Blotting of RNA ................................................27
2.3.4. DNaseI digestion of total RNA....................… 28
2.3.5. Reverse Transcription of RNA..................................… 28
2.3.6. Polymerase Chain Reaction and Reverse
Transcription Polymerase Chain Reaction
(PCR and RT-PCR)............................................................... 28
2.3.7. Quantitative RT-PCR (qRT-PCR)........29
2.3.8. Cloning of PCR products in T-vectors…………..…….. 30
2.3.9. Sequencing.................................................................. 31
2.3.10. Digestion……………..…………………………………. 31
I_______________________________________________________Table of Contents
2.3.11. Gel extraction and purification of DNA.…………….. 31
2.3.12. DNA purification by phenol/ chloroform extraction.. 32
2.3.13. Ligation………………………………………………… 32
2.4. DNA and RNA labeling method………………………………… 32
2.4.1. Random primed oligo labeling of cDNA……………... 32
2.4.2. RNA labeling methods………………………………… 33
2.4.2.1. Generation of a template………………….… 33
352.4.2.2. Radioactive RNA labeling with S
through in vitro transcription…………………….….… 34
2.4.2.3. Non radioactive RNA labeling
with DIG through in vitro transcription……….……… 34
2.5. Hybridization methods……………………………………..…….. 34
2.5.1. Radioactive Hybridization Methods…………….……. 34
2.5.1.1. Northern Blot…………………………….…… 34
2.5.1.2. Hybridization of cDNA filter……………..…... 35
2.5.1.3. In situ hybridization (ISH)…………………..… 35
2.5.2. Non-radio active hybridization methods……………... 37
2.5.2.1. Affymetrix……………………………………... 37
2.5.2.2. Fluorescent in Situ Hybridization (FISH)..… 37
2.5.2.3. Array based comparative genome
hybridization (array CGH)…………………..………… 38
2.5.2.4. Whole mount in situ hybridizaton (WISH).... 39
2.6. Protein isolation, electrophoresis, and mass spectroscopy.… 41
2.7. Histological methods…………………………………………….. 41
2.7.1. Paraffin embedding………………………………….… 41
2.7.2. Embedding for cryo sections………………………..… 42
2.7.3.Sectioning of paraffin embedded specimens………... 42
2.7.4. Cryo sectioning…………………………………………. 42
2.7.5. Vital staining of paraffin sections with
Hematoxylin & Eosin………………………………………….. 42
2.7.6. Vital staining of frozen sections with
Hematoxylin & Eosin………………………..………………… 43
2.8. Working with Bacteria culture………………….……………….. 43
2.8.1. Competent Cells……………………….………………. 43
2.8.2. Transformation………………………….……………… 44
2.9. Working with cell culture…………………………..…………….. 44
2.9.1. Standard cell culture…………………….…………….. 44
2.9.2. Transfection of adherent cells.……………………….. 44
2.9.3. Cell lines…………………..…………………………….. 45
2.9.4. EBV transformation……………………….…………… 45
2.10. Working with a Phage Library…………………….…………... 46
2.10.1. Human fetal cartilage cDNA library……….………... 46
2.10.2. Plate a phage library…………………………………… 46
2.10.3. Processing of a large number of phage clones
for sequencing ……………………………………..…………… 47
2.11. Working with Mice…………………………………….……….… 47
II_______________________________________________________Table of Contents
2.11.1. Housing mice………………………………..………… 47
2.11.2. Lmx1b knockout mice………………………..……….. 48
2.11.3. Dissection of limb buds for RNA preparation……….. 48
2.12. Immunohistochemistry…………………………………….…….. 48
2.13. Bioinformatic............................................................................ 50
2.13.1. Batched Sequence Analysis of EST sequences........ 50
2.13.2. Analysis of single sequences…………………….…… 51
2.13.3. Affimetrix normalization and analysis……………...… 52
2.14. Material………………………………………………………….… 53
2.14.1. Solutions.................................................................... 53
2.14.2. Chemicals………………………………………..……... 57
2.14.3. Miscellaneous materials………………………..……... 59
2.14.4. Kits………………………………………………..…..…. 60
2.14.5. Equipment…………………………………………..…. 61
2.14.6. Plasmids……………………………………………..… 62
2.14.7. Oligos ........................................................................ 62
2.14.7.1. Common oligos............................................ 62
2.14.7.2. Lmx1b genotyping oligos…………………… 63
2.14.7.3. Oligos to amplify inserts in pcDNA 3.1
and pcDNA 3.1/V5-HisA.............................................. 63
2.14.7.4. Oligos used for qRT-PCR……………….….. 63
2.14.7.5. Oligos used for EST clone ch11g10/
LRRC59…………………………………………..……… 66
2.14.7.6. Screening oligos for LMX1B,
TBX4, WNT4, WNT7A…………………………………. 66
2.14.7.7. Oligos used to generate ISH/WISH probes.. 68
2.14.8. Molecular weight marker……………………………… 68

3. Results..………………………………………………………………………. 69
3.1. EST project………………………………………………………… 69
3.1.1. Results from database searches…………………….… 69
3.1.2. Statistical annotation of EST sequences…………….. 71
3.1.3. Characterization of selected EST clones…………….. 78
3.1.3.1. Characterization of clone B-C12……………. 78
3.1.3.2. Characterization of clone ch11g10………….. 79
3.1.3.2.1. Knock-out model for Lrrc59………… 85
3.1.3.3. Characterization of clone ch32F03.……….… 87
3.1.4. Microarray experiment………………………………….. 91

3.1.4.1. Normalization and evaluation of microarray
data……………………………………………………….. 91
3.1.4.2. Evaluation of microarray data using qRT-
PCR………………………………………………………. 92
3.2. Lmx1b……………………………………………………………….. 94
3.2.1. Finding a downstream target of Lmx1b……………….. 95

III_______________________________________________________Table of Contents
3.2.1.1. Differentially expressed genes in fore limb
buds………………………………………………………. 97
3.2.1.2. Evaluation of micro array experiment using
qRT-PCR………………………………………………… 99
3.2.2. Evaluation of microarray experiment using WISH…… 101
3.2.2.1 Whole mount in situ hybridization for Lmx1b.. 101
3.2.2.2. Whole mount in situ hybridization for putative 101
Lmx1b downstream targets………………………….…
3.2.3. Neuropilin 2 (Nrp2)……………………………………… 102
3.3. Genitopatellar syndrome…………………………………………. 103
3.3.1. Patient # 152-01-01…………………………….. 103
3.3.2. Mutation screening…………….………………… 107
3.3.3. Array-based comparative genomic
hybridization……………………………………………... 108
3.3.4. Determination of the breakpoints………………. 110
3.3.5. Quantitative RT-PC