Abstract
ABSTRACTHundreds of hormones and ligands stimulate cyclic AMP (cAMP) signaling in different tissues through the activation of G-protein-coupled receptors (GPCRs). Although the functions and individual effectors of cAMP signaling are well characterized in many tissues, pleiotropic effects of GPCR agonists limit investigations of physiological functions of cAMP signaling in individual cell types at different developmental stages in vivo. To facilitate studies of cAMP signaling in specific cell populations in vivo, we harnessed the power of DREADD (designer receptors exclusively activated by designer drugs) technology by creating ROSA26-based knock-in mice for the conditional expression of a Gs-coupled DREADD (rM3Ds-green fluorescent protein [GFP], or “GsD”). After Cre recombinase expression, GsD is activated temporally by the administration of the ligand clozapine N-oxide (CNO). In the same allele, we engineered a CREB-luciferase reporter transgene for noninvasive bioluminescence monitoring of CREB activity. After viral delivery of Cre recombinase to hepatocytes in vivo, GsD is expressed and allows CNO-dependent cAMP signaling and glycogen breakdown. The long-term expression of GsD in the liver results in constitutive CREB activity and hyperglycemia. ROSA26-Gs-DREADD mice can be used to study the physiological effects of cAMP signaling, acute or chronic, in liver or any tissue or cell type for which transgenic or viral Cre drivers are available.
Highlights
Hundreds of hormones and ligands stimulate cyclic AMP signaling in different tissues through the activation of G-protein-coupled receptors (GPCRs)
To create a mouse line for the facile expression of GsD in any cell type, we used the same strategy as that used for the ROSA26-LSL Cre reporters in which fluorescent proteins are expressed from a CAG promoter after the Cre-mediated excision of an LSL cassette [40]
Previous work used DREADDs to elucidate the effects of G-protein-mediated signaling in the regulation of neuronal activity, glucose metabolism, and mitogenic signaling, to name a few [21, 24, 27, 28, 58]
Summary
Hundreds of hormones and ligands stimulate cyclic AMP (cAMP) signaling in different tissues through the activation of G-protein-coupled receptors (GPCRs). To facilitate studies of cAMP signaling in specific cell populations in vivo, we harnessed the power of DREADD (designer receptors exclusively activated by designer drugs) technology by creating ROSA26-based knock-in mice for the conditional expression of a Gs-coupled DREADD (rM3Ds-green fluorescent protein [GFP], or “GsD”). Random insertion transgenesis in mice to express DREADDs from tissue-specific promoters has offered an additional genetic approach but has different limitations, including unknown copy numbers, random genomic insertion sites, and a lack of flexibility in using the same animal line for expression in different tissues To circumvent these challenges and offer complementary approaches, other groups have created ROSA26-based knock-in mice with a Cre-releasable lox-stop-lox (LSL) cassette to express Gi- or Gq-coupled DREADDs in different cell types by using Cre recombinase [33, 34]. We expressed Cre recombinase in liver and primary mouse hepatocytes to characterize the ROSA26-GsD mouse line
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
