Conditional gene expression in renal collecting duct epithelial cells: use of the inducible Cre-lox system.
The renal collecting duct plays a key role in control of ion and fluid homeostasis. Genes encoding for ion transporters, hormone receptors, or regulatory proteins specifically expressed in the collecting duct are mutated in several genetic diseases with altered blood pressure. Suitable cellular models expressing genes in a conditional way should represent attractive systems for structure-function analyses and generation of appropriate physiopathological models of related diseases. However, generation of such systems remains laborious and quite inefficient. We adapted and improved a conditional Cre-lox-inducible system in the highly differentiated aldosterone-sensitive rat cortical collecting duct (RCCD2) cell line. The inducible MerCreMer recombinase allowed tight control and high levels of transgene expression, whereas flanking a selection marker with two loxP sites strongly improved the selection procedure. We have used this system to conditionally express an enhanced green fluorescent protein-tagged human mineralocorticoid receptor. In the future, this will allow structure-function analyses as well as mineralocorticoid receptor trafficking studies in these epithelial cells, which retain the features of the native collecting duct. Improvements in the conditional Cre-lox expression system have potentially wide applications in other epithelial or nonepithelial cell lines.
Ouvrard-Pascaud
Antoine
A
INSERM U478, Federative Institute of Research 02, Bichat Medical School, 75870 Paris Cedex 18, France.
Puttini
Stefania
S
Sainte-Marie
Yannis
Y
Athman
Rafika
R
Fontaine
Virginie
V
Cluzeaud
Francoise
F
Farman
Nicolette
N
Rafestin-Oblin
Marie-Edith
ME
Blot-Chabaud
Marcel
M
Jaisser
Frederic
F
D000450:Aldosterone / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004847:Epithelial Cells / Q000502:physiology*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D019426:Integrases / Q000235:genetics*; D007685:Kidney Tubules, Collecting / Q000166:cytology*; D008164:Luminescent Proteins / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D051381:Rats; D011960:Receptors, Estrogen / Q000235:genetics; D014162:Transfection; D019076:Transgenes / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics*
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011960:Receptors, Estrogen; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D000450:Aldosterone; C045073:Cre recombinase; D019426:Integrases