Quantification of p53 expression in the nervous system.
The transcription factor p53 is a short lived protein that is thought to be associated with cellular proliferation and apoptosis. In the current study, we present a protocol to measure p53 expression across both the central and peripheral nervous systems of transgenic and parental mice using the enzyme linked immuno-substrate assay (ELISA), chloramphenicol acetyl transferase reporter assay (CAT) and immunohistochemistry approaches. The profiles of the ELISA tissue data of CD1 mice were compared to the CAT assay data of the p53-promoter-driven CAT gene transgenic mice. Subsequently, high resolution immunohistochemical analysis of positive tissues in both mouse strains were evaluated. As the p53 protein is apparently subject to high turnover, the comparison of the more stable CAT data to the pan p53 ELISA assay should effectively complement each other in identifying which nervous system structures express p53. ELISA analysis alone could give ambiguous data. Immunohistochemical studies confirmed and further defined p53 expression in several regions of the nervous system. Significantly, p53 promoter-driven CAT expression was visualized in the Purkinje cells of the cerebellum and in the cornea as well as in the retina of the eye. This approach for the analysis of very short half-life proteins in the nervous system should be transferable to the study of other proteins.
Weisinger
G
G
Endocrinology Institute, Sourasky Medical Center, Tel Aviv, Israel. gary_w@tasmc.health.gov.il
Tendler
Y
Y
Zinder
O
O
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D017930:Genes, Reporter; D007150:Immunohistochemistry; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D009420:Nervous System / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D016159:Tumor Suppressor Protein p53; D015500:Chloramphenicol O-Acetyltransferase