Tetracycline-dependent gene expression in cholinergic neurons of transgenic mice [Elektronische Ressource] : a tool to study neuregulin-1 function in vivo / vorgelegt von Tobias Marc Fischer

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Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2006
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

INAUGURAL - DISSERTATION
zur
Erlangung der Doktorwürde
der
Naturwissenschaftlich-Mathematischen Gesamtfakultät
der
Ruprecht-Karls-Universität
Heidelberg















vorgelegt von
Diplom-Biologe Tobias Marc Fischer
aus Aachen


Tag der mündlichen Prüfung:






Tetracycline-dependent gene expression
in cholinergic neurons of transgenic mice—
a tool to study Neuregulin-1 function in vivo



















Gutachter:
Prof. Dr. Konrad Beyreuther
Zentrum für Molekulare Biologie
Universität Heidelberg


Dr. Cary Lai
The Scripps Research Institute
La Jolla, CA
Diese Arbeit wurde im Zeitraum von Mai 2000 bis August 2004 im Labor von Dr. Cary Lai
am The Scripps Research Institute in La Jolla, CA durchgeführt und im Zeitraum von
September 2004 bis September 2005 im Labor von Prof. Klaus-Armin Nave am Max-Planck
Institut für Experimentelle Medizin, Göttingen fortgeführt.


Acknowledgements / Danksagung:

I would like to thank Cary Lai for giving me the opportunity to work on such a challenging
project and for granting me a high degree of freedom during my work – it was fun! I would
also like to thank Janet for her excellent technical help, Isla, Jenny and Raphaëlle for their
help throughout this thesis and for making work fun and Adriane for her friendship and
cheers.

Ich möchte besonders meinen Eltern und Ingeborg Franck für ihre fortwährende
Unterstützung danken, ohne die diese Arbeit niemals möglich gewesen wäre.

Meinen Freunden, allen voran Arne, möchte ich für ihre Freundschaft danken und bei
Stephanie dafür, dass sie mich in den letzten Monaten ertragen hat, auch wenn dies nicht
immer leicht war!

Ein großer Dank gilt Markus Schwab, der mir oft während dieser Zeit wissenschaftlich und
als Freund zur Seite gestanden hat – und ohne dessen Ratschläge diese Arbeit vermutlich
nicht möglich gewesen wäre.
Bei Klaus-Armin Nave möchte ich sehr für die Möglichkeit bedanken, dass ich meine Arbeit
in seinem Labor beenden konnte.

Des Weiteren danke ich Chris, Olga, Maike, Micha, Magda, Patricia, Sven, Gudrun, Ursula,
Martin und Moritz für ihre Hilfe, guten Vorschläge und die gute Arbeitsatmosphäre.




Index

I. Summary............................................................................................................1
II. Introduction........................................................................................................2
1. The Neuregulin-1-erbB signaling network.........................................................
1.1. Structure of Neuregulin-1................................................................................... 3
1.2. Neuregulin-1 expression in the nervous system................................................. 6
1.3. Cholinergic neurons in the CNS and structural organization of the ChAT gene7
1.4. Neuregulin receptors..........................................................................................8
1.5. Neuregulin-1 function........................................................................................9
1.6. NRG1 as a regulator of myelin sheath thickness.............................................. 13
1.7. Neuregulin-2, -3 and -4 .................................................................................... 14
1.8. Transgenic approaches to study gene function in vivo..................................... 15
1.8.1. Modification of BACs by homologous recombination in bacteria .................. 15
1.8.1.1. Inducible transgenes.........................................................................................17
1.8.2. Tetracycline-regulated gene expression ........................................................... 18
1.8.2.1. Components of the tet-system .......................................................................... 19
1.9. Aims of this thesis ............................................................................................ 21
1.9.1. Aim 1: Generation of a transgenic mouse model permitting regulated gene
expression in cholinergic neurons .................................................................... 22
1.9.2. Aim 2: Generation of transgenic animals permitting the doxycycline-regulated
expression of distinct NRG1 isoforms. ............................................................ 23
III. Results..............................................................................................................24
1.1. Characterization of pBelo11 ChAT-BAC ........................................................
S1.2. Generation of pBelo11 ChAT rtTA2 -M2 ....................................................... 25
S1.3. -M2 BAC transgenics ........................... 27
S1.4. Analysis of ChAT rtTA2 -M2 BAC transgenics ............................................. 27
1.5. Alternative approach to cholinergic expression of tTA ................................... 31
1.6. Generation of RP24-70D4 ChATtTApA (‘ChATtTApA’).............................. 32
1.7. Generation of RP24-70D4 ChATtTApA BAC transgenics ............................. 33
1.8. Analysis of ChATtTApA BAC transgenics generated with linear BAC DNA 34
1.9. Analysis of the ChATtTApA#44 BAC transgenic line.................................... 35
1.10. Detection of β-galactosidase-positive Bergmann glia in ChATtTApA#44
transgenics ........................................................................................................ 38
1.11. ics generated with circular BAC DNA
.......................................................................................................................... 39
1.12. Determination of BAC copy numbers in mouse lines ChATtTApA#44 and #73 40
1.13. Whole-mount histo-chemical detection of lacZ in ChATtTApA:GFPG3 doubly
transgenic embryos........................................................................................... 42
1.14. Analysis of β-galactosidase expression in ChATtTApA#73:GFPG3 transgenic
animals.............................................................................................................. 44
2. Generation of P -bi NRG1 transgenic animals ............................................... 46 tet
2.1. Molecular cloning of NRG1 isoforms.............................................................. 46
2.2. Expression of NRG1 cDNAs in cultured cell lines.......................................... 46
2.3. Generation of P -bi NRG1 transgenics. .......................................................... 47 tet
2.4. Analysis of pNRG1typeI-lacZ (‘IgBetaBi’) transgenic animals...................... 48
2.5. III-lacZ (‘SMDFBi’) transgenic animals ................... 49
IV. Discussion........................................................................................................51
I

1. The generation of BAC transgenic mice for rtTA/tTA-dependent gene
regulation in cholinergic neurons ..................................................................... 52
1.1. The use of pBelo11 ChAT BAC to achieve cholinergic-specific gene
expression......................................................................................................... 52
S 1.2. Cholinergic expression of tTA2 through the use of the RP24-70D4 BAC..... 53
2. Generation of inducible Ptet-bi NRG1 transgenic mice................................... 54
3. Expression analysis of ChATtTApA animals using the GFPG3 mouse line ... 55
3.1. The use of circular versus linear BAC DNA for transgenesis.......................... 58
3.2. Functional consequences of the blood brain barrier for doxycycline-dependent
regulation of transgene expression ................................................................... 59
3.3. Outlook.............................................................................................................61
3.4. Future experiments...........................................................................................
3.5. Generation of additional P -bi NRG1 lines..................................................... 61 tet
4. Alternative uses of ChATtTApA mice............................................................. 62
V. Materials...........................................................................................................64
1. Chemicals and laboratory supplies
1.1. Laboratory equipment......................................................................................
1.2. Laboratory supplies..........................................................................................65
1.3. Enzymes
1.4. Antibodies........................................................................................................66
1.5. Mouse lines.......................................................................................................
1.6. Bacterial strains..................................................................................