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

Abstract

Smooth muscle alpha 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.

Figure 1. SMA-mCherry construct and characterization

(a) Diagram of BAC clone RP23-132C11 demonstrating the location of sequences substituted for SMA exon 2 creating the SMA-mCherry vector is shown. Exon 2 of the SMA locus was replaced with a myristoylated mCherry cassette at the AUG translational initiation codon to express membrane localized mCherry. (b) Standard curve to calculate copy number of SMA-mCherry lines. (c) Copy number of SMA-mCherry lines. Transgenic mice contained between 1 and 3 copies of the transgene.

Figure 2. Expression of SMA-mCherry in the developing mouse embryo

(a-d) Whole mount reporter expression in E7.5-10.5 embryos. (e,h,k,n,q,t,w,z,cc) Reporter expression (red) (f,i,l,o,r,u,x,aa,dd) SMA antibody staining (green) (g,j,m,p,s,v,y,bb,ee) and merged images of SMA-mCherry immunochemistry. Reporter expression in the developing heart and aorta at E10.5 (e-g) yolk sac (h-j) and somites (k-m) recapitulates endogenous SMA expression. Reporter expression in the E13.5 heart (n-p) aorta and esophagus (q-s) stomach and intestine smooth muscle (t-v) recapitulate endogenous SMA expression. Reporter expression in the E18.5 (w-y) heart (z-bb) lung (cc-ee) esophagus also recapitulates endogenous SMA expression. Scale bar (e-g, k-v) 100um (h-j, w-ee) scale bar 50um. A, Aorta; E, Esophagus; S, Stomach; I, Intestine.

Figure 3. SMA-mCherry expression in adult organs

(a-d) Heart (e-h) mesenteric vessels (i-l) stomach (m-p) lung (q-t) kidney (u-x) ileum of the intestine (y-bb) mammary gland (cc-ff) femoral artery. Expression was observed in (c-d, g-h, k-l, o-p, s-t, w-x, aa-bb, ee-ff) SMA-mCherry but not (a-b, e-f, i-j, m-n, q-r, u-v, y-z, cc-dd) WT controls. WT images for fluorescence were obtained with the same light intensigy and exposure time as SMA-mCherry.

Figure 4. Isolation of SMA positive cells by flow cytometry

FACS profiles of (a) WT and (b) SMA-mCherry E15.5 yolk sac cells. Cultured cells expressed mCherry (c, red) and immunostained positive for SMA (c, green).

Figure 5. Live imaging of the beating embryonic heart at E9.5

The image series is acquired from a cross of the SMA-mCherry line to the Tg(ε-globin::GFP) line labeling embryonic erythrocytes at 16 frames per second (fps) at the depth of 100 μm. (a-c) Representative frames from the image series showing different phases of the cardiac cycle. (d) Relative fluorescence intensity of the mCherry (red) and the GFP (green) as a function of time in the region marked by a circle in the panel (d).