Identification of homozygous transgenic mice by genomic real-time PCR

Schmidtke, Gunter; Gröttrup, Marcus

Identification of homozygous transgenic mice by genomic real-time PCR

Dateien

Zu diesem Dokument gibt es keine Dateien.

Datum

2008

Publikationstyp

Beitrag zu einem Sammelband

Publikationsstatus

Published

Erschienen in

MARX, Andreas, ed., Oliver SEITZ, ed.. Molecular beacons : signalling nucleic acid probes, methods, and protocols. Totowa, NJ: Humana Press, 2008, pp. 45-58. Methods in molecular biology. 429. ISBN 978-1-58829-700-6

Zusammenfassung

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.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

Zitieren

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, 2008, pp. 45-58. Methods in molecular biology. 429. ISBN 978-1-58829-700-6

BibTex

@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}
}

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    <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>
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Universitätsbibliographie

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