Identification of homozygous transgenic mice by genomic real-time PCR
Identification of homozygous transgenic mice by genomic real-time PCR
No Thumbnail Available
Files
There are no files associated with this item.
Date
2008
Editors
Journal ISSN
Electronic ISSN
ISBN
Bibliographical data
Publisher
Series
URI (citable link)
International patent number
Link to the license
EU project number
Project
Open Access publication
Collections
Title in another language
Publication type
Contribution to a collection
Publication status
Published in
Molecular beacons : signalling nucleic acid probes, methods, and protocols / Marx, Andreas; Seitz, Oliver (ed.). - Totowa, NJ : Humana Press, 2008. - (Methods in molecular biology ; 429). - pp. 45-58. - ISBN 978-1-58829-700-6
Abstract
The 26S proteasome is the executing protease of the ubiquitin-dependent degradation system. It consists of one or two 19S regulatory sub-complexes and one 20S proteolytic sub-complex (1). The 20S proteasome is a barrel-shaped cylinder which consists or four stacked rings (2). Each of the two outer rings consists of seven different alpha-subunits, whereas each of the two inner rings is formed by seven different beta-subunits (3). Only three of these beta-subunits bear a catalytically active N-terminal threonine (4,5). Under normal conditions, these are beta1 (delta), beta2 (Z), and beta5 (mb1). However, by induction of some cytokines, e.g., interferon-gamma, these subunits are exchanged against beta1i(LMP2), beta2i (Mecl1), and beta5i (LMP7) and the so-called immunoproteasome is formed (6,7). To investigate the role of LMP7 in MHC class I-restricted immunology, we decided to generate a transgenic mouse which constitutively expresses LMP7 in all tissues. To get the highest possible expression, we bread the mice to be homozygous for the transgene LMP7. These mice cannot be identified by conventional polymerase chain reaction (PCR). So far, Southern blotting was the only possible method to quantify the DNA content. Here, we describe the analysis of these mice by quantitative PCR using sequence specific fluorescence resonance energy transfer-primers to reliably detect a difference in DNA content as small as a factor of 2 or only one PCR cycle.
Summary in another language
Subject (DDC)
570 Biosciences, Biology
Keywords
Conference
Review
undefined / . - undefined, undefined. - (undefined; undefined)
Cite This
ISO 690
SCHMIDTKE, Gunter, Marcus GRÖTTRUP, 2008. Identification of homozygous transgenic mice by genomic real-time PCR. In: MARX, Andreas, ed., Oliver SEITZ, ed.. Molecular beacons : signalling nucleic acid probes, methods, and protocols. Totowa, NJ:Humana Press, pp. 45-58. ISBN 978-1-58829-700-6BibTex
@incollection{Schmidtke2008Ident-1195, year={2008}, title={Identification of homozygous transgenic mice by genomic real-time PCR}, number={429}, isbn={978-1-58829-700-6}, publisher={Humana Press}, address={Totowa, NJ}, series={Methods in molecular biology}, booktitle={Molecular beacons : signalling nucleic acid probes, methods, and protocols}, pages={45--58}, editor={Marx, Andreas and Seitz, Oliver}, author={Schmidtke, Gunter and Gröttrup, Marcus} }
RDF
<rdf:RDF xmlns:dcterms="http://purl.org/dc/terms/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#" xmlns:foaf="http://xmlns.com/foaf/0.1/" xmlns:void="http://rdfs.org/ns/void#" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/1195"> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-23T09:06:45Z</dcterms:available> <dcterms:issued>2008</dcterms:issued> <dc:creator>Gröttrup, Marcus</dc:creator> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/1195"/> <dc:language>eng</dc:language> <dc:rights>terms-of-use</dc:rights> <foaf:homepage rdf:resource="http://localhost:8080/"/> <dcterms:abstract xml:lang="eng">The 26S proteasome is the executing protease of the ubiquitin-dependent degradation system. It consists of one or two 19S regulatory sub-complexes and one 20S proteolytic sub-complex (1). The 20S proteasome is a barrel-shaped cylinder which consists or four stacked rings (2). Each of the two outer rings consists of seven different alpha-subunits, whereas each of the two inner rings is formed by seven different beta-subunits (3). Only three of these beta-subunits bear a catalytically active N-terminal threonine (4,5). Under normal conditions, these are beta1 (delta), beta2 (Z), and beta5 (mb1). However, by induction of some cytokines, e.g., interferon-gamma, these subunits are exchanged against beta1i(LMP2), beta2i (Mecl1), and beta5i (LMP7) and the so-called immunoproteasome is formed (6,7). To investigate the role of LMP7 in MHC class I-restricted immunology, we decided to generate a transgenic mouse which constitutively expresses LMP7 in all tissues. To get the highest possible expression, we bread the mice to be homozygous for the transgene LMP7. These mice cannot be identified by conventional polymerase chain reaction (PCR). So far, Southern blotting was the only possible method to quantify the DNA content. Here, we describe the analysis of these mice by quantitative PCR using sequence specific fluorescence resonance energy transfer-primers to reliably detect a difference in DNA content as small as a factor of 2 or only one PCR cycle.</dcterms:abstract> <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/> <dcterms:title>Identification of homozygous transgenic mice by genomic real-time PCR</dcterms:title> <dc:creator>Schmidtke, Gunter</dc:creator> <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-23T09:06:45Z</dc:date> <dc:contributor>Schmidtke, Gunter</dc:contributor> <dcterms:rights rdf:resource="https://rightsstatements.org/page/InC/1.0/"/> <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/> <dcterms:bibliographicCitation>Publ. in: Molecular beacons : signalling nucleic acid probes, methods, and protocols / ed. by Andreas Marx and Oliver Seitz. (Methods in molecular biology ; 429). Totowa, NJ : Humana Press, 2008, pp. 45-58</dcterms:bibliographicCitation> <dc:contributor>Gröttrup, Marcus</dc:contributor> </rdf:Description> </rdf:RDF>
Internal note
xmlui.Submission.submit.DescribeStep.inputForms.label.kops_note_fromSubmitter
Examination date of dissertation
Method of financing
Comment on publication
Alliance license
Corresponding Authors der Uni Konstanz vorhanden
International Co-Authors
Bibliography of Konstanz
Yes