Derivation of β cell–specific HNF-4α knockout mice. (A) HNF-4α^loxP/loxP mice in which exon 2 was flanked by loxP sites were bred to Ins.Cre-transgenic mice expressing Cre recombinase under control of the rat insulin promoter. The resulting HNF-4α^loxP/+; Ins.Cre offspring were then mated with HNF 4α^loxP/loxP homozygotes to obtain HNF-4α^loxP/loxP; Ins.Cre mutants and their littermate controls: HNF-4α^loxP/+, HNF-4α^loxP/loxP, and HNF-4α^loxP/+; Ins.Cre. (B) Primers 1, 2, and 3 (red, blue, and green in A) were used for PCR genotyping of isolated islets from HNF-4α^loxP/loxP; Ins.Cre and HNF-4α^loxP/loxP mice. In the absence of Cre, amplification by primers 1 and 2 results in a 620-bp product. Cre-mediated excision of exon 2 results in a 450-bp product amplified by primers 1 and 3. Quantification of the bands shows that deletion occurs in approximately 70% of all islet cells (note that non–β cells make up 20–30% of the islet cell numbers). (C) Concordant with the results in B, mRNA levels of HNF-4α were reduced by 63% in mutant islets, as determined by quantitative PCR using primers specific to exon 2. *P < 0.05; n = 3 per group. (D and E) Immunostaining of pancreatic sections from adult control (D) and mutant (E) mice using an antibody against HNF-4α indicates that the number of β cells expressing HNF-4α protein is reduced by approximately 85–90% (arrow) in the mutant mouse. Non–β cells in the islet mantle still express HNF-4α protein (arrowhead) in the mutant mouse. Thus, HNF-4α is deleted efficiently and specifically in pancreatic β cells. Magnification, ×200.