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Targeted Disruption of Gnas in Embryonic Stem Cells¹

Division of Endocrinology and Metabolism (W.F.S., M.A.L.), Department of Medicine and Division of Developmental Genetics (K.J.R., A.M.L., J.D.G.), Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: William F. Schwindinger, Division of Endocrinology and Metabolism, Johns Hopkins University School of Medicine, Ross 863, 720 Rutland Avenue, Baltimore, Maryland 21205. E-mail: wschwind{at}welchlink.welch.jhu.edu

Mutations in the gene encoding the stimulatory G protein of adenylyl cyclase (G_s) are present in subjects with Albright hereditary osteodystrophy, a syndrome of characteristic developmental defects and, in some patients, resistance to multiple hormones that stimulate cAMP accumulation (pseudohypoparathyroidism type Ia). As the first step in generating a model of Albright hereditary osteodystrophy, the gene encoding G_s (Gnas) was disrupted in mouse embryonic stem (ES) cells by homologous recombination. Northern blot analysis and immunoblot analysis demonstrated that steady-state levels of G_s messenger RNA and G_s protein in targeted ES cells were approximately 50% of levels in untargeted ES cells. In response to 10 µM forskolin and to various concentrations of isoproterenol (0.1–3.0 µM), cAMP accumulation was reduced in the G_s knockout ES cell lines, relative to wild-type ES cells and to five of six ES cell lines with randomly integrated targeting vector. These results support the role of G_s haploinsufficiency in reducing the ability of hormones to generate cAMP in subjects with pseudohypoparathyroidism type Ia. The targeted disruption of Gnas in mouse ES cells establishes an in vitro system for further studies of the role of G_s and cAMP coupled signal transduction in differentiation and development.

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