Targeted disruption of Gnas in embryonic stem cells

William F. Schwindinger, Kimberly J. Reese, Ann M Lawler, John D. Gearhart, Michael A. Levine

Research output: Contribution to journalArticle

Abstract

Mutations in the gene encoding the stimulatory G protein of adenylyl cyclase (Gα(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 (Gα(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 (Gα(s)) messenger RNA and (Gα(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 (Gα(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 (Gα(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 (Gα(s)) and cAMP coupled signal transduction in differentiation and development.

Original languageEnglish (US)
Pages (from-to)4058-4063
Number of pages6
JournalEndocrinology
Volume138
Issue number10
DOIs
StatePublished - 1997

Fingerprint

Embryonic Stem Cells
Pseudohypoparathyroidism
Hormones
Haploinsufficiency
Cell Line
Homologous Recombination
Colforsin
Isoproterenol
GTP-Binding Proteins
Adenylyl Cyclases
Northern Blotting
Genes
Signal Transduction
Messenger RNA
Mutation
Proteins
Mouse Embryonic Stem Cells
Albright's hereditary osteodystrophy

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Schwindinger, W. F., Reese, K. J., Lawler, A. M., Gearhart, J. D., & Levine, M. A. (1997). Targeted disruption of Gnas in embryonic stem cells. Endocrinology, 138(10), 4058-4063. https://doi.org/10.1210/en.138.10.4058

Targeted disruption of Gnas in embryonic stem cells. / Schwindinger, William F.; Reese, Kimberly J.; Lawler, Ann M; Gearhart, John D.; Levine, Michael A.

In: Endocrinology, Vol. 138, No. 10, 1997, p. 4058-4063.

Research output: Contribution to journalArticle

Schwindinger, WF, Reese, KJ, Lawler, AM, Gearhart, JD & Levine, MA 1997, 'Targeted disruption of Gnas in embryonic stem cells', Endocrinology, vol. 138, no. 10, pp. 4058-4063. https://doi.org/10.1210/en.138.10.4058
Schwindinger WF, Reese KJ, Lawler AM, Gearhart JD, Levine MA. Targeted disruption of Gnas in embryonic stem cells. Endocrinology. 1997;138(10):4058-4063. https://doi.org/10.1210/en.138.10.4058
Schwindinger, William F. ; Reese, Kimberly J. ; Lawler, Ann M ; Gearhart, John D. ; Levine, Michael A. / Targeted disruption of Gnas in embryonic stem cells. In: Endocrinology. 1997 ; Vol. 138, No. 10. pp. 4058-4063.
@article{55d3b762bca84c1498c90b44796744d5,
title = "Targeted disruption of Gnas in embryonic stem cells",
abstract = "Mutations in the gene encoding the stimulatory G protein of adenylyl cyclase (Gα(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 (Gα(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 (Gα(s)) messenger RNA and (Gα(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 (Gα(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 (Gα(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 (Gα(s)) and cAMP coupled signal transduction in differentiation and development.",
author = "Schwindinger, {William F.} and Reese, {Kimberly J.} and Lawler, {Ann M} and Gearhart, {John D.} and Levine, {Michael A.}",
year = "1997",
doi = "10.1210/en.138.10.4058",
language = "English (US)",
volume = "138",
pages = "4058--4063",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "The Endocrine Society",
number = "10",

}

TY - JOUR

T1 - Targeted disruption of Gnas in embryonic stem cells

AU - Schwindinger, William F.

AU - Reese, Kimberly J.

AU - Lawler, Ann M

AU - Gearhart, John D.

AU - Levine, Michael A.

PY - 1997

Y1 - 1997

N2 - Mutations in the gene encoding the stimulatory G protein of adenylyl cyclase (Gα(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 (Gα(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 (Gα(s)) messenger RNA and (Gα(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 (Gα(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 (Gα(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 (Gα(s)) and cAMP coupled signal transduction in differentiation and development.

AB - Mutations in the gene encoding the stimulatory G protein of adenylyl cyclase (Gα(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 (Gα(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 (Gα(s)) messenger RNA and (Gα(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 (Gα(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 (Gα(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 (Gα(s)) and cAMP coupled signal transduction in differentiation and development.

UR - http://www.scopus.com/inward/record.url?scp=0030886333&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030886333&partnerID=8YFLogxK

U2 - 10.1210/en.138.10.4058

DO - 10.1210/en.138.10.4058

M3 - Article

C2 - 9322912

AN - SCOPUS:0030886333

VL - 138

SP - 4058

EP - 4063

JO - Endocrinology

JF - Endocrinology

SN - 0013-7227

IS - 10

ER -