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Publié par | ruprecht-karls-universitat_heidelberg |
Publié le | 01 janvier 2008 |
Nombre de lectures | 26 |
Langue | English |
Poids de l'ouvrage | 13 Mo |
Extrait
Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto‐Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
presented by
Master Sci. Ming‐Fei Lang
born in: Heilongjiang, China
Oral‐examination:: ................................................
1
A transgenic strategy to define SLCO1C1‐expressing
structures during brain development
Referees: Prof. Dr. Günther Schütz
Prof. Dr. Markus Schwaninger
2
Dedicated to my parents
3
Hiermit erkläre ich, daß ich die vorliegende Dissertation selbst verfaßt und mich
dabei keiner anderen als der von mir ausdrücklich bezeichneten Quellen und
Hilfen bedient habe. Des Weiteren erkläre ich, daß ich an keiner anderen Stelle
ein Prüfungsverfahren beantragt oder die Dissertation in dieser oder einer
anderen Form bereits anderweitig als Prüfungsarbeit verwendet oder einer n Fakultät als Dissertation vorgelegt habe.
Heidelberg,
Ming‐Fei Lang
4
Acknowledgements
Acknowledgements
The work in this dissertation was performed in the lab of Prof. Markus Schwaninger. I would like to take
this opportunity to thank,
Prof. Markus Schwaninger for the interesting projects, for his supervision, support and for providing
excellent working environment throughout my study.
Prof. Günther Schütz for his positive input and scientific support.
Dr. Armin Schneider (Sygnis Pharma AG, Heidelberg) for providing the data of PGRP‐S mRNA
CNSKOaccumulation in WT and RelA mice.
Jana Hroudova for providing most part of the data on the expression pattern of Cre.
Dr. Ioana Inta for the RcCMV‐p65 plasmid and technical support at the beginning of my study.
Waleed Barakat for providing cDNAs from TM‐BBB4 cells.
Dr. Marion Schölzke for providing cDNAs from cultured neural stem cells.
Dr. Stefan Berger for technical support on BAC recombineering and providing EL250 cells and pConst
and pERT2 plasmids.
Dr. Hai‐Kun Liu for technical support on BAC recombineering, providing Cre and β‐gal antibodies and
helpful discussions.
Dr. David Engblom, Dr. Milen Kirilov and Dr. Ying Wang for their technical support on BAC
recombineering.
Prof. Roman Dziarski (Indiana University, Gary, USA) for providing the PGRP‐S knockout mice and
allowing me to perform some experiments in his lab.
Melanie Neubert for translating the summary into German.
And all the other members of AG Schwaninger that were not mentioned above for their support and
discussions.
The Max‐Planck Society for financial support in the first year of this dissertation.
The GK791/2 of the University of Heidelberg for accepting me and providing me with various
opportunities to improve myself.
5
Summary
Summary
Thyroid hormones (THs) are important for brain development. Maternal hypothyroxinemia, low
maternal thyroxine levels, is a major cause of defects in fetal brain development. In adults, thyroxine
enters brain through the thyroxine transporter SLCO1C1, which is expressed in endothelial cells of the
blood‐brain barrier (BBB) and in epithelial cells of the choroid plexus. Till now, SLCO1C1 is the only
known transporter that mediates the entry of thyroxine into the adult brain. However, the expression
pattern of SLCO1C1 during development was unknown.
To characterize the expression of SLCO1C1 in development, a transgenic approach was employed by
bacterial artificial chromosome (BAC) recombineering. Sequences of Cre recombinase (constitutive) or
Cre recombinase fused to a mutated estrogen receptor ligand binding domain (inducible) were inserted
into the Slco1c1 locus on a BAC by homologous recombination. The modified BACs were subsequently
injected into pronuclei to generate mice carrying the Slco1c1‐driven Cre, which is expressed either
constitutively or in an inducible manner.
In the transgenic mice, both Cre and Slco1c1 transcripts could be detected by RT‐PCR only in the brain.
Mice from the constitutive line showed Cre expression in BBB and choroid plexus according to
immunohistochemistry. This localization was confirmed by crossing mice with a reporter mouse line. In
addition, neurons in many brain regions (such as cortical layer 2/3, the hippocampus and olfactory
bulbs) expressed Cre during brain development, since Cre activity could not be detected in adult
neurons but Cre‐mediated recombination was found in the neurogenic zones at E14 and E18. In the
inducible line, no Cre activity in the adult neurons was found, which also confirmed an early neuronal
expression of Slco1c1‐Cre. The neuronal expression of Cre in development indicates a role of thyroxine
in the SLCO1C1‐expressing neurons. Hypothyroxinemia induced by propyl‐thio‐uracil (PTU) decreased
the number of cortical