Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle α‐actin gene

Abstract

Smooth muscle α actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells (vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a membrane localized cherry red fluorescent protein (mCherry), driven by the full-length SMA promoter and intronic sequences. We determined that the founders and F1 progeny of five independent lines contain 1-3 copies of the mCherry substituted BAC vector. Furthermore, we characterized the expression of SMA-mCherry in relation to endogenous SMA in the embryo and in adult tissues, and found that the transgenic reporter in each line recapitulated endogenous SMA expression at all time points. We were also able to isolate SMA expressing cells from embryonic tissues using fluorescence activated cell sorting (FACS). We demonstrated that this marker can be combined with other vital fluorescent reporters and it can be used for live imaging of embryonic cardiodynamics. Therefore, these transgenic mice will be useful for isolating live SMA-expressing cells via FACS and for studying the emergence, behavior and regulation of SMA-expressing cells, including vascular smooth muscle cells and pericytes throughout embryonic and postnatal development.

Dynamic responses of endothelial cells to changes in blood flow during vascular remodeling of the mouse yolk sac

“…However, an analysis of the variance of the density values from E8.5-10.5 showed a decrease in variance at later stages ( Fig. 1J; P=0.004, Bartlett test), perhaps as a response to vessel stabilization or to smooth muscle cell investment (Armstrong et al, 2010). We also observed a slightly lower median value at E10.5 (P=0.0081, Kruskal-Wallis test; Fig.…”

“…Although it is clear that vessel remodeling is regulated by changes in hemodynamic force (Lucitti et al, 2007), it is unclear how such forces could alter the morphology and diameter of vessels. In mature vessels, where the endothelium is surrounded by contractile smooth muscle cells, blood flow can increase vessel diameter through muscle relaxation, but in the embryo smooth muscle cells are not detected surrounding vessels until after E9.5, when changes in diameter are already evident (Armstrong et al, 2010). This suggests that changes in diameter are likely to be regulated directly at the level of the EC.…”

Recombineering of BAC DNA for the Generation of Transgenic Mice

“…4c,f, arrowhead). Heart development and function can also be monitored and measured using these mice, as previously reported (Armstrong et al 2010). …”

“…1a). Our immunohistochemistry on cultured cells selected for mCherry expression by Fluorescent Activated Cell Sorting (FACS) showed a distinctly different membrane localization of our mCherry reporter compared to the cytoskeletal staining pattern of SMA indicating we had avoided generating a SMA-mCherry fusion (Armstrong, Larina et al 2010). …”

“…We adopted this method to measure copy number in our SMA-mCherry transgenic lines (Armstrong, Larina et al 2010), and found that the level of expression of the reporter correlated with BAC copy number, which is useful for the comparative evaluation of the mouse lines.…”