**4.1 Experimental design**

In our studies, we were interested in generating a reporter mouse line to monitor the expression of Smooth Muscle--Actin (SMA) in vivo and track the fate of cells expressing this gene. Transcriptional control of the SMA locus had been well defined in previous studies (Mack and Owens 1999; Mack, Thompson et al. 2000). Using that information, we decided to replace exon 2 (the first coding exon of SMA) and 50bp distal of the 3' end of exon 2, with a myristoylated mCherry fluorescent reporter (Shaner, Campbell et al. 2004). Our rationale for this design was to a) insert the reporter in a manner where the first codon of the reporter replaced the first codon of SMA, and b) to avoid generating a fusion protein by removing the exon 2 splice site donor (Fig. 1a).

Our targeting construct consisted of a mCherry expression sequence with a myristoylation sequence upstream of a PGK-neomycin resistance cassette (Fig. 1a). The PGK-neomycin resistance cassette was flanked at the 5' and 3' end by FRT recombination sites. Inclusion of the PGK-neomycin resistance cassette facilitated selection of positive recombinant clones by kanamycin (Fig. 1b). Inclusion of the FRT recombination sites allowed for subsequent excision of the PGK-neomycin resistance cassette by Flpe recombinase, thus eliminating any unwanted transcription effects of the PGK promoter on the reporter (Fig. 1c). The targeting construct ended with a SV40 polyadenylation sequence to enable efficient transcription (Fig. 1a).

Fig. 1. Experimental design for the generation of a Smooth-Muscle--Actin (SMA) reporter construct. (a) The endogenous SMA locus and linear targeting construct. (b) The SMA locus correctly targeted by recombineering. (c) The final reporter construct following excision of PGK-neomycin selection cassette.

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)**.**
