Visualization of whole-mount skeletal expression patterns of LacZ reporters using a tissue clearing protocol.
Gene targeting or trapping constructs that utilize the lacZ gene encoding beta-galactosidase activity to trap promoter expression have become an increasingly important way to disrupt gene function and monitor gene expression. A number of genes targeted in this way have revealed both expected and unexpected developmental abnormalities of the skeleton. The use of X-gal staining to monitor gene expression in developing skeletal structures is hampered in these mutants because, during the critical latter stages of mouse embryonic development, visualization is hindered by the opacity of overlying soft tissue. Here, we report the development of a reliable method to clear exogenous tissue in late-stage embryos and neonates that still preserves skeletal X-gal staining patterns. This protocol reveals (i) specific cell staining in localized regions of developing bone and cartilage in two different genetic models and (ii) that the intensity of X-gal staining is consistent with the level of expression of lacZ. We conclude that this protocol accurately reflects both the specificity and intensity of expression and will facilitate the analysis of mouse skeletal development.
Kawaguchi
Jitsutaro
J
Centre for Genome Research, University of Edinburgh, UK.
Wilson
Valerie
V
Mee
Patrick J
PJ
D000818:Animals; D001842:Bone and Bones / Q000196:embryology / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D006579:Heterozygote; D006720:Homozygote; D007763:Lac Operon; D051379:Mice; D008957:Models, Genetic; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics; D015238:Sulfotransferases / Q000235:genetics
D015238:Sulfotransferases; C106358:heparan-sulfate 2-sulfotransferase; D010744:Phosphoric Monoester Hydrolases