**4.2 Isolation of BAC DNA**

26 Bacterial Artificial Chromosomes

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

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

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

**4.1 Experimental design** 

by removing the exon 2 splice site donor (Fig. 1a).

sequence to enable efficient transcription (Fig. 1a).

PGK-neomycin selection cassette.

Isolation of BAC DNA can be achieved using a standard alkaline lysis phenol/chloroform extraction followed by alcohol precipitation. However, these types of BAC DNA preparations are subject to contamination by genomic DNA. Column purification of BAC DNA, such as that achieved using NucleoBond BAC 100 kit by Clontech, will yield more pure and less degraded BAC DNA. With either procedure, the removal of fragmented linear genomic DNA can be achieved using ExoV exonuclease or increasing lysis of the bacterial wall using lysozyme, as needed. However, large BAC DNA sequences are susceptible to shearing, so one must take care during preparation so as not to degrade the intact BAC DNA.
