P200
DEVELOPMENT OF A NOVEL MURINE MODEL OF NEPHRONOPHTHISIS
Eley, L, Hynes, A, Simms, R, Miles, C, Sayer, J
Institute of Human Genetics, Newcastle University
Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, is an important
genetic cause of end-stage renal disease in children and young adults. Recent genetic
advances have allowed positional cloning and identification of several NPHP genes (NPHP19) involved in nephronophthisis. NPHP6 encodes nephrocystin-6, a centrosomal protein.
Here we create and characterise a novel murine model of nephronophthisis, carrying a
mutation in the Nphp6 gene.
A murine ES cell line containing an Nphp6 “gene trap” was identified using database
searches. Re-sequencing confirmed this cell line harboured a β-galactosidase reporter gene (βgeo) inserted into the endogenous Nphp6 locus leading to a truncating mutation. ES cells were
cultured before injecting into murine blastocysts to create chimaeric mice. Chimeras were
bred to produce viable, healthy heterozygous mutant mice. The β-geo reporter gene, being
placed under transcriptional control of the Nphp6 regulatory elements, allowed determination
of the spatio-temopral expression pattern of Nphp6 in heterozygous mutant mice, using X-gal
staining of tissue in adult and embryos. In Nphp6+/- animals, specific X-gal staining
representative of Nphp6 expression was observed in a wide range of tissues (including kidney
and cerebellum) at various embryological time points. Interestingly, in renal tissues prominent
Nphp6 expression was noted at the corticomedullary junction.
Heterozygous mutant mice have been intercrossed to produce mice homozygous for
the Nphp6 truncating mutation. Importantly, Nphp6-/- animals (homozygous for the gene trap
Nphp6lacZ) exhibit cystic kidney disease which commences from birth and progresses through
the first 3 months of life. Histological examination reveals that these cysts are collecting duct
cysts, staining positively for aquaporin-2 and -3.
The development of Nphp6+/- mice harbouring a reporter gene will allow a detailed
expression pattern of Nphp6 to be established, and correlated with antibody studies. Tubular
expression of Nphp6 throughout kidney development is noteworthy. Strong expression at the
corticomedullary junction of the kidneys is relevant as the phenotype of nephronophthisis
includes cysts specifically at this region. The homozygote mutant mouse will provide an
excellent model to investigate the mechanisms involved in cyst formation and the effects of
therapeutic agents on cyst expansion both in vivo and in vitro.