TARGETED DISRUPTION OF GNAS IN EMBRYONIC STEM-CELLS

Mutations in the gene encoding the stimulatory G protein of adenylyl c yclase (G alpha(s)) are present in subjects with Albright hereditary o steodystrophy, 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 en coding G alpha(S) (Gnas) was disrupted in mouse embryonic stem (ES) ce lls by homologous recombination. Northern blot analysis and immunoblot analysis demonstrated that steady-state levels of G alpha(s) messenge r RNA and G alpha(s) protein in targeted ES cells were approximately 5 0% of levels in untargeted ES cells. In response to 10 mu M forskolin and to various concentrations of isoproterenol (0.1-3.0 mu M), cAMP ac cumulation was reduced in the G alpha(s) knockout ES cell lines, relat ive to wild-type ES cells and to five of six ES cell lines with random ly integrated targeting vector. These results support the role of G al pha(s) haploinsufficiency in reducing the ability of hormones to gener ate cAMP in subjects with pseudohypoparathyroidism type Ia. The target ed disruption of Gnas in mouse ES cells establishes an in vitro system for further studies of the role of G alpha(s) and cAMP coupled signal transduction in differentiation and development.