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Fig. 1. Generation of Brn1 knockout mice and histological changes in their kidneys at birth. (A) Representation of the wild-type allele, targeting vector and targeted allele of Brn1. The Brn1 open reading frame is shown as a black box. The locations of the external 3' and internal 5' probes are indicated. NEO, neomycin-resistance gene driven by phosphoglycerate kinase gene promoter; DTA, diphtheria toxin A-chain gene; E, EcoRI; B, BamHI; X, XhoI; N, NotI; A, ApaI. (B) Western blot analysis of kidney extracts from newborn Brn1 mutants. The specific band of Brn1 detected by a polyclonal antiserum is reduced in Brn1^+/- mice and absent from Brn1^-/- mice. (C) Electrophoresis mobility-shift assay (EMSA) using brain extracts derived from newborn Brn1 mutants. Cell extracts of Brn1-transfected NIH 3T3 cells (3T3-Brn1) served as a Brn1 protein control. Lysates of P19 cells treated with retinoic acid (P19-RA) were used as a Brn2 protein-positive control. (D,E) Staining of the kidney medulla derived from Brn1^+/+ (D) and Brn1^-/- (E) mice with Hematoxylin and periodic acid-Schiff (PAS). The collecting ducts (CD) in Brn1^-/- kidneys are comparable with those of the Brn1^+/+ kidney. The lops of Henle (HL), however, are absent from the Brn1^-/- kidney. Interstitial cells (IC) are prominent in the Brn1^-/- kidney in comparison with the wild-type kidney. (F,G) Cortices of Brn1^+/+ (F) and Brn1^-/- (G) kidneys stained with Hematoxylin and Eosin. No significant differences in cortex morphology were observed between Brn1^+/+ and Brn1^-/- kidneys. Gl, glomerulus. Scale bar: 50 µm.

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