Mutations in the gene (GNASI) encoding the stimulatory G protein of adenylyl cyclase (Gas) have been identified in subjects with Albright hereditary osteodystrophy (AHO), an inherited disorder with characteristic developmental defects and in some patients resistance to hormones that stimulate cAMP production (pseudohypoparathyroidism type la). We intend to establish a transgenic mouse model of AHO by targeted disruption of the GNAS1 gene. A 6kb BamHI fragment of the GNASI gene, extending from 2 kb upstream of the initiator ATG to the middle of exon 2, was isolated from a 129SVJ genomic library. A targeting vector was constructed by replacing a 500 bp fragment extending from the NcoI site at the initiator ATG to an Nrul site in intron 1 with the neomycin resistance gene (neoR). ES cells (J1) were transfected by electroporafion, and selected in 250 ug/ml G418. Southern blot analysis with oligonucleotide probes upstream of the 5' BamHl site showed an additional band of the expected size in 3 of 142 colonies, indicating targeted disruption of the GNAS-1 gene. Total RNA and plasma membranes were prepared from one of the Ga, knockout ES cell line and 3 other ES cell lines with randomly integrated targeting vector. Immunoblot analysis revealed a 5316% decrease in the steady state level of Gas protein, and Northern blot analysis revealed a 43+8% decrease in Gas, mRNA, in the Gas knockout ES cells relative to the other 3 clones. cAMP accumulation was measured in confluent cultures of ES cells in 24- well dishes. In response to 10-5 M Forskolin and to various doses of lsoproterenol, cAMP accumulation was reduced in the Gas knockout ES cell line, relative to 6 ES cell lines with random integration of targeting vector. Under these same conditions, cAMP accumulation in each of the 3 Gas, knockout cell lines was also reduced relative to the wild type ES cell line (J1). Two of the three Gas knockout cell lines have abnormal karyotypes and are not suitable for injection into mouse embryos. Injection of the one remaining Gas knockout ES cell line into C57BL/6J mouse embryos yeilded 17 chimeric mice (7 males and 10 females). To date, none of the 43 pups with agouti coats surviving into adulthood carry the targeted GNASI allele. However 1 of 15 pups that died in the first day of life carried the targeted allele. These data raise the possibility that disruption of GNASI in mice leads to perinatal lethal phenotype. To be certain that this does not reflect an unrecognized defect in this one properly targeted clone, 4 additional properly targeted clones have been idenfified; two of these have been shown to have a normal karyotype and have been injected into mouse embryos.
Showing the most recent 10 out of 1014 publications
