Identification of homozygous transgenic mice by genomic real-time PCR.
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.
Schmidtke
Gunter
G
Department of Biology, University of Konstanz, Konstanz, Germany.
Groettrup
Marcus
M
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006579:Heterozygote; D006720:Homozygote; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D009097:Multienzyme Complexes; D011506:Proteins; D012333:RNA, Messenger; D004247:DNA; C077436:LMP7 protein; D046988:Proteasome Endopeptidase Complex; C486213:Psme1 protein, mouse