Abstract P1001: Comprehensive “Tool Box” For Delineating Adrenergic Receptor Function

Abstract

Sympathetic regulation of the cardiovascular system is required to maintain cardiac output and vascular tone in response to varied physiological stressors. This includes the release norepinepherine and epinephrine. Nor/epinephrine acts on nine, evolutionarily conserved, adrenergic, G-protein coupled receptors. Three alpha 1 ( Adra1 ), 3 alpha 2 ( Adra2 ), and 3 beta ( Adrb ) receptors. Understanding the distribution and cell-specific function of each of these receptors may lead to new targeted therapies for cardiovascular disease. To delineate the relative roles of each of the 9 adrenergic receptors, we are taking a genetic approach to study these receptors in mice; specifically, by using the Cre-loxP system to target the varied receptors in a tissue-specific manner. Unfortunately, there is a dearth of such tools to study these receptors. We therefore are in the process of generating three sets of tools for each of the 9 adrenergic receptors using the CRISPR-Cas9 method. 1. Adrenergic receptor floxed mice. 2. Adrenergic receptor reactivation (transcriptional blocking) mice and 3. Adrenergic receptor Cre mice. Thus far, we have generated Adra1a , Adra1b , Adra1d , Adra2a , Adrb2 , and Adrb3 floxed mice by inserting loxP sites flanking at least one exon near the 5’ end of the gene. The floxed mice were crossed to a germline-Cre (CMV-Cre), generating mice with whole-body loss of function alleles. Complementing the conditional loss of function alleles, we are also generating reactivation alleles by inserting a loxP-flanked transcriptional blocking cassette (TB) in each of the 9 endogenous adrenergic receptor genes. These TB alleles will allow us to reactivate endogenous expression of each adrenergic receptor in a Cre-dependent manner in an otherwise global knockout background. This model will allow us to identify tissues which are sufficient for the function of each adrenergic receptor. Finally, the ires-Cre models will allow for targeting of each adrenergic receptor-expressing cells with genetic reporters, and physiological studies using chemo and/or optogenetics. These new tools will assist in identifying the site of action and physiological function of various receptor subtypes and provide mechanistic insights into adrenergic receptor signaling.