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Use of an internal ribosome entry site for bicistronic expression of Cre recombinase or rtTA transactivator
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
1999 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 27, no 6, 1552-1554 p.Article in journal (Refereed) Published
Abstract [en]

Conditional gene targeting depends on tissue and time specificity of recombination events. Endogenous promoters are often used to drive various transgenic constructs. To avoid the problems associated with reconstituting a specific expression pattern in transgenic animals by this method, we tested the internal ribosome entry site of the encephalomyocarditis virus, to enable linkage of the Cre recombinase or rtTA trans-activator to 3' untranslated ends of endogenous genes. Here we report that these constructs function effectively in COS cells. The data suggest that these cassettes will be appropriate for 3' targeting of mouse genes.

Place, publisher, year, edition, pages
1999. Vol. 27, no 6, 1552-1554 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-89676DOI: 10.1093/nar/27.6.1552PubMedID: 10037820OAI: oai:DiVA.org:uu-89676DiVA: diva2:161354
Available from: 2002-03-20 Created: 2002-03-20 Last updated: 2017-12-14Bibliographically approved
In thesis
1. 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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