Mx1‐Cre mediated Rgs12 conditional knockout mice exhibit increased bone mass phenotype

Abstract

Regulators of G-protein Signaling (Rgs) proteins are the members of a multigene family of GTPase-accelerating proteins (GAP) for the Galpha subunit of heterotrimeric G-proteins. Rgs proteins play critical roles in the regulation of G protein couple receptor (GPCR) signaling in normal physiology and human diseases such as cancer, heart diseases and inflammation. Rgs12 is the largest protein of the Rgs protein family. Some in vitro studies have demonstrated that Rgs12 plays a critical role in regulating cell differentiation and migration; however its function and mechanism in vivo is largely unknown. Here, we generated a floxed Rgs12 allele (Rgs12flox/flox) in which the exon 2, containing both PDZ and PTB_PID domains of Rgs12, was flanked with two loxp sites. By using the inducible Mx1-cre and Poly I:C system to specifically delete Rgs12 at postnatal 10 days in interferon-responsive cells including monocyte and macrophage cells, we found that Rgs12 mutant mice had growth retardation with the phenotype of increased bone mass. We further found that deletion of Rgs12 reduced osteoclast numbers and had no significant effect on osteoblast formation. Thus, Rgs12flox/flox conditional mice provide a valuable tool for in vivo analysis of Rgs12 function and mechanism through time- and cell-specific deletion of Rgs12.

Regulators of G protein signaling 12 promotes osteoclastogenesis in bone remodeling and pathological bone loss

“…To assess the role of Rgs12 in OC differentiation and function in vivo, we crossed Rgs12 fl/fl mice 25 with either CD11b-Cre 22 or Mx1-Cre 23 transgenic lines to generate Rgs12 conditional knockout mice (CD11b;Rgs12 fl/fl and Mx1;Rgs12 fl/fl , respectively). In Mx1;Rgs12 fl/fl mice, Rgs12 was deleted in hematopoietic cells, including OCs, by injecting poly I:C at postnatal days 10, 12, and 14.…”

“…To define the role of Rgs12 in vivo, we generated Rgs12 conditional knockout mice. 25 Mx1-Cre targets not only OC precursor cells but also multiple hematopoietic cell types, including megakaryocytes, lymphocytes, mast cells, and monocytemacrophages. To specifically understand the role of Rgs12 in OC lineage in vivo, we also generated a specific conditional knockout model with CD11b-Cre for deleting Rgs12 more exclusively in the OC lineage cells at a relatively early and developmental stage 22 to facilitate understanding of Rgs12 in OC differentiation and function.…”

“…Methodology for generation of Rgs12 fl/fl , Mx1-Cre, and CD11b-Cre mice have been previously described. 22,23,25 To generate Rgs12 fl/+ ;CD11b-Cre mice, Rgs12 fl/fl mice were crossed with CD11b-Cre transgenic mice. To generate Rgs12 fl/fl ;CD11b-Cre mice (referred to as CD11b;Rgs12 fl/fl ), female Rgs12 fl/fl mice were crossed with male Rgs12 fl/+ ;CD11b-Cre mice.…”

“…24 Genotyping of mice was performed by PCR of proteinase K-digested tail or spleen DNA as we have previously done. 25 The Cre transgene was detected using primer CreF (5′-CCTGGAAAATGCTT CTGTCCGTTTGCC-3′) and primer CreB (5′-GGCGCGGCAACACCATTTTT-3′). The deleted, floxed, and wild-type alleles were genotyped using three primers: S (5′-CAGTTATTGGAAACTATCTCATGAC-3′); M (5′-TCCCCAAGCCTCTTACTTCA-3′); and W (5′-CACCGCACACACAAAATAAA TATCA-3′).…”

“…Fixed femur from Rgs12 mutant and Cre control mice (n = 4) were analyzed and 3D reconstitution was used to determine bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) as described. 25,51,52 Histology and histomorphometric analyses. Mice femurs and tibias were excised, fixed for 24 h in 10% natural buffered formalin, and decalcified in 10% EDTA for 1-2 weeks at 4°C.…”