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Transgenic expression of Cre recombinase from the tyrosine hydroxylase locus
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
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2004 (English)In: Genesis, ISSN 1526-954X, E-ISSN 1526-968X, Vol. 40, no 2, 67-73 p.Article in journal (Refereed) Published
Abstract [en]

Catecholaminergic neurons are affected in several neurological and psychiatric diseases. Tyrosine hydroxylase (TH) is the first, rate-limiting enzyme in catecholamine synthesis. We report a knockin mouse expressing Cre-recombinase from the 3'-untranslated region of the endogenous Th gene by means of an internal ribosomal entry sequence (IRES). The resulting Cre expression matches the normal pattern of TH expression, while the pattern and level of TH are not altered in the knockin mouse. Crossings with two different LacZ reporter mice demonstrated Cre-mediated genomic recombination in TH expressing tissues. In addition, LacZ was found in some unexpected cell populations (including oocytes), indicating recombination due to transient developmental TH expression. Our novel knockin mouse can be used for generation of tissue-specific or general knockouts (depending on scheme of crossing) in mice carrying genes flanked by loxP sites. This knockin mouse can also be used for tracing cell lineages expressing TH during development.

Place, publisher, year, edition, pages
2004. Vol. 40, no 2, 67-73 p.
Keyword [en]
3' Untranslated Regions, Adrenal Glands/metabolism, Animals, Brain Chemistry/immunology, Electroporation, Female, Genes; Reporter, Heterozygote, Immunohistochemistry, Integrases/*metabolism, Lac Operon, Male, Mice, Mice; Inbred C57BL, Mice; Transgenic, Recombination; Genetic, Research Support; Non-U.S. Gov't, Stem Cells, Tissue Distribution/genetics, Transgenes, Tyrosine 3-Monooxygenase/*genetics, Viral Proteins/*metabolism, beta-Galactosidase/metabolism
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-91751DOI: 10.1002/gene.20065PubMedID: 15452869OAI: oai:DiVA.org:uu-91751DiVA: diva2:164583
Available from: 2004-04-21 Created: 2004-04-21 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Role of Bone Morphogenetic Proteins for Catecholaminergic Neurons in Vivo: Use of the Tyrosine Hydroxylase Locus for Cell-Specific inactivation of Signal Transduction
Open this publication in new window or tab >>Role of Bone Morphogenetic Proteins for Catecholaminergic Neurons in Vivo: Use of the Tyrosine Hydroxylase Locus for Cell-Specific inactivation of Signal Transduction
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Members of the Transforming Growth factor-β (TGF-β) superfamily and its subclass Bone Morphogenetic Proteins (BMP) play important roles for nervous system development.

In order to study the BMP role for catecholaminergic neurons in vivo, we generated three knock-in mice, expressing the transgenes specifically in the targeting cells.

Two genetic modifications result in expression of dominant negative (dn) BMP receptors (BMPRII and ALK2). The tissue-specific expression was achieved by the transgene insertion into 3’- untranslated region of the endogenous gene for tyrosine hydroxylase (TH), the first enzyme in catecholamine biosynthesis. An Internal Ribosome Entry site (IRES) preceded inserted cDNAs, allowing for functional bicistronic mRNA production. While almost no defects in Th-IRES-dnALK2, the Th-IRES-dnBMPRII mouse demonstrated declined levels of catecholamines, including dopamine in the striatum. Losses of midbrain dopaminergic neurons (MDN) might cause the effect. Additionally, intermediate lines of these mice, preserving a neo-cassette, oriented opposite to the locus transcription, demonstrate dramatic decrease of catecholamine level, hence, represent models for rare catecholamine-deficiency diseases, including L-DOPA-responsive dystonia.

The third mouse, expressing in the same way Cre-recombinase (Th-IRES-Cre), represents a tool for catecholaminergic cell-limited deletion of any gene, which has to be flanked by loxP sites. Besides TH-positive areas, unexpected sites of Cre-recombination were identified, indicating regions of transient TH expression. Surprising recombination in oocytes opens a possibility to use our mouse as a general Cre-deletor.

Using TH-IRES-Cre mouse we generated tissue-specific knockout mice for two BMP signal transducers: Smad1 and Smad4 (also crucial for TGF-β). While no phenotype in Smad1 knockout, TH-IRES-Cre/Smad4 mouse revealed several defects including decreased level of striatal dopamine.

These results demonstrate a positive role of BMPs for MDN fate in vivo. Generated mice represent a tool-box for comprehensive study of the BMP function in catecholaminergic neurons. This study is of potential interest for understanding some aspects of Parkinson’s disease.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 74 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1350
Keyword
Neurosciences, Tyrosine Hydroxylase, Bone Morphogenetic Proteins (BMP), Transforming Growth Factor-β (TGF-β), tissue-specific knockout, Parkinson's disease, Neurovetenskap
National Category
Neurology
Identifiers
urn:nbn:se:uu:diva-4258 (URN)91-554-5964-1 (ISBN)
Public defence
2004-05-13, B21, BMC, Husargatan 3, Uppsala, 10:15
Opponent
Supervisors
Available from: 2004-04-21 Created: 2004-04-21Bibliographically approved
2. New Conditional Gene Targeting Methods: For Studying Neurotrophic Mechanisms in Selected Neuronal Populations
Open this publication in new window or tab >>New Conditional Gene Targeting Methods: For Studying Neurotrophic Mechanisms in Selected Neuronal Populations
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Powerful techniques to manipulate the mouse genome have had a great impact on our understanding of biology. The major drawback with conventional transgenic methods is that the genetic alteration will be present in every cell of the animal, from conception and onwards. Many genes are normally expressed in several different organs and at different times and an effect observed in the transgenic mouse could therefore be derived from various tissues or be a developmental consequence. Furthermore, some mutations will result in a lethal phenotype and prevent investigations on the adult gene function. This is a particular problem when studying the brain since it is not yet fully developed at birth. Consequently, a conditional methodology is required, where precise chromosomal alterations can be achieved in restricted tissues at chosen times in the living animal. This issue has been successfully tackled during the last decade. Conditional gene targeting is today a fact.

This thesis demonstrates that it is possible to use an internal ribosomal entry sequence for tissue-specific direction of conditional gene targeting tools. General cassettes were constructed and shown to work in cells. Moreover, a knock-in transgenic mouse expressing the Cre recombinase in catecholaminergic neurons was made. This strain will offer possibilities to study genetic mechanisms in dopamine and noradrenaline producing cells and to build mouse models for common human diseases involving such neurons.

Furthermore, another transgenic mouse was created having the Cre recombinase under tight tetracycline-regulated control. This strain can mediate inducible genetic recombination in brain neurons and be completely silenced by feeding the mice the antibiotic doxycycline. Moreover, by altering the time-point of doxycycline administration, chromosomal manipulations can be achieved in different neuronal patterns. This transgene is a general tool that can be combined with any mouse having floxed genes and also with lines producing tetracycline transactivators in other locations.

Finally, constructs aiming at conditional manipulation of the nerve growth factor gene and targeting of sensory neuronal populations are described.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 68 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1126
Keyword
Neurosciences, Neurovetenskap
National Category
Neurology
Research subject
Developmental Neurosciences
Identifiers
urn:nbn:se:uu:diva-1774 (URN)91-554-5247-7 (ISBN)
Public defence
2002-04-12, Sal B21, Biomedicinska centrum, Uppsala, 10:15
Opponent
Available from: 2002-03-20 Created: 2002-03-20 Last updated: 2013-05-17Bibliographically approved

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Kylberg, AnnikaEbendal, Ted

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