Figure 1. Generation of mice expressing LRb^L985.

(A) Cartoon of signaling by LRb showing the activation of specific signals by individual tyrosine phosphorylation (pY) sites. (B) Targeting strategy for generation of the Lepr^L985 allele. Shown is the targeting vector containing the LRb-specific exon 18b with the mutation of Tyr985 (which also generates a novel HindIII site) (exon18b-L985) plus Neo and thymidine kinase (TK) cassettes for positive and negative selection, respectively. Recombination in ES cells replaces the wild-type exon 18b with exon 18b-L985 and the Neo cassette, to mediate physiologic expression of the mutant LRb from the endogenous Lepr gene, as previously described (26). (C) Southern blot using a Lepr locus probe, demonstrating correct targeting of the locus in +/+, l/+, and l/l mice. M, marker. (D) qPCR for hypothalamic LRb mRNA expression in +/+ and l/l mice. (E) Hypothalamic detection of STAT3(PY) in +/+ and l/l mice. Age- and sex-matched animals were treated i.p. with vehicle or leptin for 30 minutes. Brains were processed for immunohistochemical detection of STAT3(PY) in the hypothalamus; representative sections showing the ARC and ventromedial hypothalamic nucleus (VMH) are shown. For reference, position of the third cerebral ventricle (3V) is shown. Original magnification, ×20.