Ex Vivo Embryonic Kidney Culture
The Mouse Metanephric Organ Culture Cyst Growth Assay (1,2) allows an analysis of the early cyst-forming process during embryonic kidney development in genetic mouse models of PKD and mouse models expressing conditional alleles using segment specific or inducible Cre recombinase constructs or reporter constructs to track the expression of PKD associated proteins. Briefly, embryonic kidneys are cultured for 4-5 days and undergo metanephric induction, ureteric bud branching, and tubule elongation. Cystic dilations form in PKD mutant kidneys and to a lesser extent in wild type kidneys in a cyclic AMP-dependent process involving NKCC1 and CFTR-dependent chloride ion secretion. Cyst growth in culture is monitored daily and digitally recorded for quantification. Tubule segments are identified by lectin staining. Kidneys can be treated with diffusible small molecules or peptides to manipulate cyst formation and enlargement. RNA and protein expression and histology can be used to analyze the cyst forming process. PKD RRC can provide expert advice and detailed protocols for the assay or can offer experimental support on a 1) fee for service basis or as a 2) collaborative arrangement if there is mutual interest.
1. Magenheimer, B.S., St. John, P.L., Isom, K.S., Abrahamson, D.R., De Lisle, R.C., Wallace, D.P., Maser, R.L., Grantham, J.J., and Calvet, J.P. (2006) Early embryonic renal tubules of wild-type and PKD kidneys respond to cAMP stimulation with CFTR/NKCC1-dependent cystic dilation. J. Am. Soc. Nephrol. 17, 3424-3437. PMID: 17108316. This paper was the first to show cyst formation in PKD mutant embryonic kidneys in organ culture. It has been cited >60 times.
2. Maser, R.L., Magenheimer, B.S., Calvet, J.P. (2019) Metanephric organ culture. Methods Cell Biol. 153, 169-183. PMID: 31395378. This is a recent, detailed methods chapter that provides lists of reagents and step-by-step protocols for dissection, culturing, treatment and analysis of metanephric cultures.
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