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New Conditional Gene Targeting Methods: For Studying Neurotrophic Mechanisms in Selected Neuronal Populations
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
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 [en]
Neurosciences
Keyword [sv]
Neurovetenskap
National Category
Neurology
Research subject
Developmental Neurosciences
Identifiers
URN: urn:nbn:se:uu:diva-1774ISBN: 91-554-5247-7 (print)OAI: oai:DiVA.org:uu-1774DiVA: diva2:161358
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
List of papers
1. Use of an internal ribosome entry site for bicistronic expression of Cre recombinase or rtTA transactivator
Open this publication in new window or tab >>Use of an internal ribosome entry site for bicistronic expression of Cre recombinase or rtTA transactivator
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-89676 (URN)10.1093/nar/27.6.1552 (DOI)10037820 (PubMedID)
Available from: 2002-03-20 Created: 2002-03-20 Last updated: 2017-12-14Bibliographically approved
2. Transgenic expression of Cre recombinase from the tyrosine hydroxylase locus
Open this publication in new window or tab >>Transgenic expression of Cre recombinase from the tyrosine hydroxylase locus
Show others...
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.

Keyword
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:nbn:se:uu:diva-91751 (URN)10.1002/gene.20065 (DOI)15452869 (PubMedID)
Available from: 2004-04-21 Created: 2004-04-21 Last updated: 2017-12-14Bibliographically approved
3. Timing the doxycycline yields different patterns of genomic recombination in brain neurons with a new inducible Cre transgene
Open this publication in new window or tab >>Timing the doxycycline yields different patterns of genomic recombination in brain neurons with a new inducible Cre transgene
2002 (English)In: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 68, no 2, 248-253 p.Article in journal (Refereed) Published
Abstract [en]

We have developed a transgenic mouse expressing the Cre recombinase under control of a tetracycline-responsive promoter. Using a CamKIIalpha-driven tTA transgenic strain and a lacZ reporter mouse, we obtained the expected neuronal pattern of recombination in the olfactory lobe, cortex, striatum, hippocampus and Purkinje cells. Moreover, recombination can be completely abolished by feeding the mice doxycycline in their drinking water. We also show that it is possible to get a different pattern of recombination by changing the timing of the doxycycline-mediated shutdown of Cre expression. By starting the doxycycline treatment at birth, we restrict recombination to striatum only. This approach should be applicable to other inducible transgenic strains, thus increasing the number of available tissue-specific patterns for conditional knockouts. Also, our tetO-Cre transgene can be combined with any of the increasing number of tetracycline transactivator transgenic strains to direct specifically inducible genomic recombination to several areas of the brain.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-89678 (URN)10.1002/jnr.10213 (DOI)11948670 (PubMedID)
Available from: 2002-03-20 Created: 2002-03-20 Last updated: 2017-12-14Bibliographically approved
4. Identification of a chicken homologue in the Brn-3 subfamily of POU-transcription factors
Open this publication in new window or tab >>Identification of a chicken homologue in the Brn-3 subfamily of POU-transcription factors
1997 In: Developmental Brain Research, Vol. 100, 169-82 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89679 (URN)
Available from: 2002-03-20 Created: 2002-03-20Bibliographically approved

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