Quantitative genotyping of mouse brain-specific PEX13 gene disruption by real-time PCR.
The Cre/loxP-system has become an invaluable tool for the generation of tissue-specific gene disruption in mice. However, because Cre recombinase excision of individual genes can be variable, an accurate and sensitive method is necessary to determine the ultimate level of gene disruption. The analysis of gene disruption is particularly difficult for tissue that has been fixed for (immuno)histochemical analysis with paraformaldehyde. Here, we describe a simple, rapid and cost effective method for measurement of gene disruption using quantitative real-time PCR, through application to the analysis of PEX13 gene disruption in a brain-specific PEX13 mouse mutant. We show that this general protocol is suitable for both normal and paraformaldehyde-fixed tissue.
Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Brisbane, Australia.
C Catharina
CC
Müller
Jamie P
JP
Nourse
Tam H
TH
Nguyen
Denis I
DI
Crane
D000818:Animals; D015139:Blotting, Southern; D001921:Brain / Q000378:metabolism*; D005838:Genotype; D019426:Integrases / Q000235:genetics; D007381:Intermediate Filament Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics; D064231:Nestin; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D007381:Intermediate Filament Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; C491106:Pex13 protein, mouse; C045073:Cre recombinase; D019426:Integrases