Functional analysis of Loqs and R2D2 in the network of small RNA silencing pathways [Elektronische Ressource] / Julia Verena Hartig

Julia Verena Hartig

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 21
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München





Functional analysis of Loqs and R2D2 in the network of small
RNA silencing pathways














Julia Verena Hartig
aus
Grünstadt


2010 Erklärung


Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 von Herrn Prof. Dr. Klaus Förstemann betreut.


Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.


München, am 25.6.10


........................................................
Julia Verena Hartig





Dissertation eingereicht am 27.4.2010
1. Gutachter: Prof. Dr. Klaus Förstemann
2. Gutachter: Prof. Dr. Michael Sattler

Mündliche Prüfung am 18.6.2010
2 Contents


Contents
1 ZUSAMMENFASSUNG .............................................................................................................................. 6
2 SUMMARY ............................................................................................................................................... 8
3 INTRODUCTION...... 10
3.1 CLASSES OF SMALL RNAS ........................................................................................................................... 10
3.1.1 Micro RNAs (miRNAs) ...................... 10
3.1.2 Exogenous siRNAs (exo-siRNAs) ...................................... 12
3.1.3 Endogenous siRNAs (endo-siRNAs).................................................................. 13
3.1.4 Piwi-interacting RNAs (piRNAs) ....... 13
THE SIGNIFICANCE OF SMALL RNA SILENCING ................................. 14 3.2
3.2.1 Roles of small RNA silencing pathways ........................................................................................... 14
3.2.2 Transposable elements .................................................... 15
3.3 THE PROBLEM OF PATHWAY SPECIFICITY ........................................................................ 16
3.3.1 dsRBPs as specificity factors for small RNA sorting ......... 16
3.3.1.1 Properties of double-stranded RNA binding domains ............ 16
3.3.1.2 R2D2 ....................................................................................................................................................... 17
3.3.1.3 Loquacious .............. 17
3.3.2 Sorting and specificity problems...................................... 17
3.3.2.1 Sorting between miRNAs and exo-siRNAs .............................................................................................. 17
3.3.2.2 Sorting between miRNAs and endo-siRNAs............................ 18
3.3.2.3 Sorting between endo-siRNAs and exo-siRNAs ...................... 18
3.4 DROSOPHILA GENETICS .............................................................................................................................. 18
GFP-based cell culture reporter systems ......................... 18 3.4.1
3.4.2 Transgenic flies ................................ 20
3.4.2.1 Embryo injection and transgenic fly lines ............................................................................................... 20
3.4.2.2 The UAS/Gal4 expression system ........... 20
3.5 SMALL RNA SILENCING SYSTEMS IN OTHER ORGANISMS ................... 22
4 SPECIFIC AIMS OF THIS THESIS ............................................................................................................... 24
5 MATERIALS AND METHODS ................... 25
5.1 MATERIALS ............................................................................................................. 25
5.1.1 Laboratory hardware ...................................................... 25
5.1.2 Analysis software ............................................................................................ 25
5.1.3 Laboratory chemicals ...................... 26
5.1.4 Radiochemicals ................................................................ 27
5.1.5 Enzymes ........................................................................... 28
5.1.5.1 General enzymes .................................... 28
5.1.5.2 Polymerases ........................................................................................................... 28
5.1.5.3 Restriction enzymes ............................... 28
5.1.6 Kits ................................................................................................................................................... 28
5.1.7 Other materials 29
5.1.8 Plasmids in laboratory stock ........... 30
5.1.9 Cells ................................................................................................................................................. 31
5.1.9.1 Bacterial stocks ....... 31
5.1.9.2 Cell lines.................................................. 32
5.1.10 Fly stocks ..................................................................................................... 32
5.1.11 PCR primers . 33
5.1.11.1 Cloning .................................................... 33
5.1.11.2 Sequencing ............................................................................. 37
3 Contents


5.1.11.3 qPCR ....................................................................................................................................................... 37
5.1.11.4 Test-PCR ................................................................................. 38
5.1.11.5 RACE 38
5.1.11.6 Mapping P-element insertions in transgenic flies .................................................. 38
5.1.11.7 Sequencing primer for pUASP-trangenic flies......................................................... 39
5.1.12 Media .......................................................................... 40
5.1.12.1 Bacterial stocks ....................................................................................................... 40
5.1.12.2 Cell culture.............. 40
5.1.13 Fly food........ 41
5.1.14 Antibodies ................................................................................................................................... 41
5.1.14.1 Primary antibodies . 41
5.1.14.2 Secondary antibodies ............................................................................................. 42
5.1.15 Stock solutions and commonly used buffers ............................................... 42
5.2 METHODS ................................ 46
5.2.1 Molecular cloning ............................................................................................................................ 46
5.2.1.1 Primer design for cloning of dsRBDs ....................................... 46
5.2.1.2 Amplification of DNA sequences by Polymerase Chain Reaction (PCR) ................. 46
5.2.1.3 Agarose gel electrophoresis ................................................................................................................... 47
5.2.1.4 Specific digestion of DNA by restriction endonucleases ......... 47
5.2.1.5 Ligation of vector with insert DNA ......... 47
5.2.1.6 Bacterial transformation ........................................................................................................................ 48
5.2.1.7 Test for correct transformants by colony-PCR 48
5.2.1.8 Preparation of plasmid DNA ................... 48
5.2.1.9 DNA sequencing ..................................................................................................................................... 48
5.2.1.10 3´-RACE PCR analysis of the loqs-RD variant .......................... 48
5.2.2 Methods of Drosophila S2 cell culture ............................. 49
5.2.2.1 Maintenance .......................................................................................................................................... 49
5.2.2.2 Depletion of individual genes by RNAi in cell culture ............. 49
5.2.2.3 Selection of clonal cell lines .................... 51
5.2.2.4 Storage of cells in liquid nitrogen ........................................................................................................... 51
5.2.3 Protein analysis ............................................................... 51
5.2.3.1 Protein extraction ... 51
5.2.3.2 Co-immunoprecipitation ........................................................................................................................ 52
5.2.3.3 Immunoblotting for detection of proteins ............................. 52
5.2.3.4 α-Loqs-PD-specific antibody production ................................ 53
5.2.3.5 Dot blot ................................................................................................................................................... 53
5.2.4 RNA analysis .... 53
5.2.4.1 RNA extraction ....................................................................................................................................... 53
5.2.4.2 Northern Blotting ... 53
5.2.4.3 Analysis of mRNA levels by Polymerase Chain Reaction ........ 54
5.2.5