Novel approaches to transgenesis in the teleost medaka (Oryzias latipes) [Elektronische Ressource] / vorgelegt von Clemens Grabher

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2003
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 13 Mo

Extrait

Novel Approaches to Transgenesis in the
Teleost Medaka (Oryzias latipes)
Clemens GrabherINAUGURAL – DISSERTATION
zur
Erlangung der Doktorwürde
der
Naturwissenschaftlich-Mathemathischen Gesamtfakultät
der
Ruprecht-Karls-Universität
Heidelberg
vorgelegt von
Mag. Clemens Grabher
aus St. Gallen
Tag der mündlichen Prüfung: 15.05.2003Thema
Novel Approaches to Transgenesis
in the Teleost Medaka (Oryzias latipes)
Gutachter: PD Dr. Jochen Wittbrodt
PD Dr. Dirk-Henner LankenauDISSERTATION
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the
Rupero-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
presented by
Mag. Clemens Grabher
born in St. Gallen
Oral examination: 15.05.2003Table of Contents
Table of Contents
Table of Contents........................................................................................................ 1
Summary..................................................................................................................... 3
Introduction................................................................................................................ 5
1. Medaka - a Model System for Vertebrate Developmental Genetics............... 6
2. Transgenesis in Fish..................................................................................... 8
2.1 Methods of Transgene Delivery................................................................................................8
2.1.1 Retroviral Infection...............................................................................................................9
2.1.2 Electroporation......................................................................................................................9
2.1.3 Particle Bombardment........................................................................................................10
2.1.4 Embryonic Stem (ES) cells ................................................................................................11
2.1.5 Nuclear Transfer .................................................................................................................11
2.1.6 Microinjection.....................................................................................................................12
2.2 General Fates of Injected Transgenic DNA............................................................................13
2.2.1 Immediate Fate of Injected DNA – Transient Expression................................................14
2.2.2 Late Fate of Injected DNA – Stable (Genomic) Expression.............................................16
2.3 Strategies to Improve Transgenesis by Microinjection..........................................................17
2.3.1 Nuclear Localization Signal (NLS)....................................................................................18
2.3.2 Restriction Endonuclease Mediated Integration (REMI)..................................................19
2.3.3 Boundary Regions ..............................................................................................................19
2.3.4 Transposable Elements.......................................................................................................20
2.3.4.1 RNA Elements .........................................................................................................21
2.3.4.2 DNA Elements.........................................................................................................22
2.4 Novel Strategies to Improve Transgenesis by Microinjection...............................................24
2.4.1 The I-SceI Meganuclease ...................................................................................................25
2.4.2 The Sleeping Beauty Transposon System..........................................................................27
Aims of the Thesis..................................................................................................... 30
Results ...................................................................................................................... 32
3. Application of the I-SceI Meganuclease in Medaka .................................... 32
3.1 Co-injection of Reporter Gene and I-SceI Meganuclease Leads to Uniform
Promoter Dependent GFP Expression in G0..........................................................................32
3.2 Generation of Germ Line Transmitting Fish by I-SceI Meganuclease Co-injection ............35
3.3 Improvement in G0 Transgene Expression is Not Linked to a Nuclear Targeting
Activity of the Meganuclease..................................................................................................39
4. The SB Transposon System in Oryzias Latipes ........................................... 41
4.1 Application of the SB System Results in Increased Numbers of G0 Embryos
Uniformly Expressing GFP ....................................................................................................41
4.2 Establishment of Stable Transgenic Lines Using the SB System ..........................................44
4.3 Genomic Integration of Single or Multiple Copies................................................................45
4.4 High Frequency Generation of Spatially and Temporally Restricted Expression
Patterns in F1 Progeny.............................................................................................................48
Discussion................................................................................................................. 54
5. I-SceI Meganuclease and the SB Transposon System Mediate Highly
Efficient Transgenesis in Fish..................................................................... 54
5.1 Future Aspects .........................................................................................................................61
5.1.1 Gene Targeting ...................................................................................................................61
1Table of Contents
5.1.2 Genetic Transposition.........................................................................................................64
5.1.3 The GAL4/UAS System in Medaka....................................................................................66
Appendix to Discussion............................................................................................. 67
7. Appendix A: Application of the I-SceI Meganuclease in Medaka ............... 67
7.1 Establishing a Heat-Shock Inducible GAL4 Driver Line Reveals a Major Toxicity
Upon Over-expression.............................................................................................................67
8. Appendix B: The SB Transposon System in Oryzias Latipes ...................... 69
8.1 Repeated Germ Line Transposition Does Not Occur Upon Genetic
Transposase Induction.............................................................................................................69
Materials and Methods ............................................................................................. 72
9. Materials .................................................................................................... 72
9.1 Buffers and Media ...................................................................................................................72
9.2 Enzymes and Standards...........................................................................................................74
9.3 Kits ...........................................................................................................................................74
9.4 Chemicals.................................................................................................................................75
9.5 Bacteria ....................................................................................................................................75
9.6 Vectors .....................................................................................................................................76
9.7 Equipment................................................................................................................................77
9.8 Additional Materials................................................................................................................78
9.9 Medaka Stocks.........................................................................................................................78
10. Methods ..................................................................................................... 78
10.1 Isolation of Genomic DNA from Adult Fish..........................................................................78
10.2 Southern Blot Hybridisation....................................................................................................79
10.3 Sequencing...............................................................................................................................80
10.4 Microinjections ........................................................................................................................80
10.4.1 Meganuclease......................................................................................................................80
10.4.2 Sleeping Beauty.....................................................................................