RNA interference as a tool for functional neurogenetics and the role of microRNAs in brain function [Elektronische Ressource] / Peter Weber

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• Biologie

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 14 Mo

Extrait

Technische Universi T ä T München
Lehrstuhl für e ntwicklungsgenetik
rnA interference as a tool for
functional neurogenetics and the role
of micrornAs in brain function
Peter Weber
v ollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
e rnährung, Landnutzung und Umwelt der Technischen Universität München zur e rlangung
des akademischen Grades eines
Doktors der n aturwissenschaften
genehmigten Dissertation.
v orsitzender: Univ.-Prof. Dr. A. Gierl
Prüfer der Dissertation: 1. Univ.-Prof. Dr. W. Wurst
2. apl. Prof. Dr. J. Adamski
Die Dissertation wurde am 30.11.2009 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für e rnährung, Landnutzung
und Umwelt am 18.02.2010 angenommen.Index
Index
1. Introduction: Elucidation and applications of RNA interference (RNAi) 1
1.1. rnA in the spotlight 1
1.2. h istorical perspective on rnAi 2
1.2.1. Posttranscriptional gene silencing (PTGs) in plants 2
1.2.2. Te discovery of rnAi 3
1.3. e lucidation of the molecular mechanism behind rnAi 4
1.3.1. Discovery of the rnAi pathway 4
1.3.2. Genetic screens for mutants lacking rnA induced gene silencing 5
1.3.3. characterization of Dicer and risc 5
1.3.4. Action of rnA-dependent rnA polymerase (rdrP) in amplifcation
and transition 9
1.3.5. s ystemic spreading of rnAi 11
1.4. Biological functions of rnAi 12
1.4.1. Alterations in chromatin structure 12
1.4.2. v iral defence in plants 14
1.4.3. Transposon silencing 14
1.4.4. rnAi in development 15
1.5. rnAi as a tool 15
1.5.1. rnAi in mammalian cells 16
1.5.2. r ules for the design of functional sirnAs 17
1.5.3. v ector systems for expression of shrnAs 17
1.5.4. in vivo rnAi in mice 19
1.5.5. rnAi in the adult mouse brain 21
1.6. Micro rnAs (mirnAs) as novel functional genetic units 23
1.6.1. identifcation and cloning of mirnAs 23
1.6.2. Biogenesis and mode of action 24
1.6.3. Functions of micrornAs 26
1.6.4. e xpression studies of mirnAs 27
1.6.5. s pecifc roles of mirnAs in the brain 30
1.7. Aim of the thesis 34
2. Materials and methods 36
2.1. Materials 36
2.1.1. chemicals 36
2.1.2. e nzymes 38
iiIndex
2.1.3. n ucleotides und nucleic acids 38
2.1.4. Kits and other expendable items 39
2.1.5. Devices and equipment 40
2.2. Media and basic bufers 41
2.3. Oligonucleotides 43
2.3.1. DnA oligonucleotides 43
2.3.2. rn 44
2.3.3. LnA modifed oligonucleotides 45
2.4 v ectors: 45
2.4.1. Plasmids 45
2.4.2. v iral vectors 46
2.4.3. riboprobes for in situ hybridization 46
2.5. Antibodies 47
2. 6. Organisms 47
2.6.1. Bacterial strains 47
2.6.2. e ucaryotic cells 47
2.6.3. Animals 47
2.7. Molecular biology methods 48
2.7.1. Bacterial culture 48
2.7.2 DnA techniques 50
2.7.3. rn 55
2.7.4. Protein techniques 64
2.7.5. cell culture techniques 69
2.8. Animal experiments 73
2.8.1. Mouse housing and breeding 73
2.8.2. s tereotactic surgery 74
2.8.3 injection of sirnAs 74
2.8.4. v iral injection 75
2.8.5. Perfusion 75
2.8.6. clearing of brain tissue 75
2.8.7. Parafn embedding of brains 76
2.8.8. s ectioning of brains 76
2.8.9. Generation of transgenic mouse lines 77
2.9 Microscopy and image acquisition 77
2.9.1. Brightfeld, darkfeld, and fuorescence microscopy 77
2.9.2. Ultramicroscopy 78
2.9.3. image processing 78
iiiIndex
2.10. s tatistics, bioinformatics and computational analysis 78
2.10.1. s tatistics for pairwise group comparisons 78
2.10.2. s tatistics and analysis of mirnA arrays 78
2.10.3. DnA alignment, BLAsT and digital vector construction 79
3. Results 80
3.1. establishment of a novel rnAi expression vector 80
3.1.1. introduction: rnA polymerase i (Pol i) 80
3.1.2. rnAi vector construction 81
3.1.3. Functional characterisation of the novel vector: silencing of reporter
constructs 82
3.1.4. Molecular characterization of the novel vector 84
3.2. In vivo rnAi in mouse brain 88
3.2.1. s tereotactic injections into the mouse brain 89
3.2.2. n on-viral delivery of sirnAs 89
3.2.2. v iral vectors for rnAi delivery 90
3.3. r egulation of mirnAs by neuronal activity 95
3.3.1. h ypothesis: involvement of mirnAs in dendritic regulation of protein
translation upon neuronal activity 95
3.3.2. induction of strong neuronal activity in mouse brains by treatment with
kainic acid 96
3.3.3. Analysis of diferential mirnA expression by macro arrays 96
3.4. e xpression studies of mirnAs 106
3.4.1. catalog of mirnAs expressed in mouse hippocampus 106
3.4.2. Development of an in situ hybridization technology for mirnAs 109
3.4.3. e xpression analysis of candidate mirnAs with putative relevance to brain
development or function 111
4. Discussion 123
4.1. Generation of a novel Pol-i based rnAi vector 123
4.2. In vivo rnAi 124
4.3. r egulation of mirnAs by neuronal activity 128
4.4. e xpression studies of mirnAs 131
5. Summary 138
6. Abbreviations 140
7. References 143
8. Acknowledgements 174
iv. Introduction
1. Introduction: Elucidation and applica-
tions of RNA interference (RNAi)
1.1. RNA in the spotlight
Te e importance tance of of rnArnA moleculesmolecules hashas beenbeen underestimatedestimated forfor decadesdecades asas compareded toto theirtheir
prominent sibling DnA. rnA has only been believed to be a transient messenger in transferring
DnA’s information into protein.
But during the last years rnA biology has been one of the most innovative felds in science since
the discovery of small rnA species with important functions on regulation of gene expression,
cell diferentiation and stabilisation of the genome’s integrity struck a new path in fundamental
1-3biology and added a new link to our understanding of life .
Te phenomenon of sequence specifc gene silencing induced by double stranded rnA (dsrnA)
is called rnA interference (rnAi). When dsrnA is introduced into cells, genes with sequence
homology to this dsrnA are suppressed. Tis phenomenon was newly discovered when experi -
4ments with sense and antisense rnA-mediated gene inhibition were accidently combined .
Te impact on the scientifc community was tremendous and scientifc publications on this
topic have been arising since then (Figure 1).
Te view on rnA has been revolutionized, similar to some other great discoveries in life sciences
such as DnA as molecule of heredity, the immune system of mammals, and prions. Terefore
5the “science” journal quoted rnAi as the most important scientifc topic in 2002 and in the
4,500
QuantumMechanics
4,000
RNAi
3,500
3,000
2,500
2,000
1,500
1,000
500
0
Figure 1: RNAi as novel highly dynamic research feld. On the left side the cover of the last “Science” issue of the year 2002 is shown
5in which the editors termed small RNAs as the most important topic of that year . On the right side a bar plot illustrates the number
of publications listed in Web of Science® under the topic “RNA interference” or “RNAi” from the year 1995 to 2008 compared to the
publication count with the topic “quantum mechanics” in the same time

1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008. Introduction
year 2006 Andrew Z. Fire and c raig c. Mello have been awarded with the n obel Prize in Physi-
ology or Medicine “for their discovery of rnA interference - gene silencing by double-stranded
rnA”. Additionally v ictor Ambros, David Baulcombe, and Gary r uvkun won thethe Albertt Lask-Lask-
er Basic Medical r esearch Award in 2008 “for discoveries that revealed an unanticipated world
6of tiny rnAs that regulate gene function in plants and animals” .
r evealing the biology underlying rnAi in diferent species, the unexpected observation of gene
silencing and other phenomena were integrated into a more comprehensive view on the role of
rnA in the regulation of gene expression.
1.2. Historical perspective on RNAi
in the middle of the 1980s, a novel technique was established utilizing antisense rnA to inhibit
7gene function in cultured murine cells . DnA expression constructs were generated by excis -
ing the protein coding sequence of a cloned gene and this sequence was reinserted in reverse
orientation in relation to the promoter. Tese constructs showed inhibition either injected or
transfected into cells.
8injection of in vitro transcribed antisense rnA into Drosophila embryos resulted in specifc
down regulation of the targeted genes. Tis antisense rnA technique also worked in transgenic
9 10organisms and in an inducible manner . Tereby the antisense rnA was believed to hybrid -
ize to the mrnA by Watson c rick base pairing and thus prohibiting mrnA translation of this
specifc gene.
in the course of time, rnAi technology has been used widely for evaluating gene function in all
genetic model organisms. Te most prominent example for this application is the so called Flavr
® s avr transgenic tomato (1988). Tis tomato was generated by the calgene start-up company
and became the frst engineered food to gain FDA approval in 1994. in these