**6. Utility of BAC transgenics**

30 Bacterial Artificial Chromosomes

and cellular specificity. Not only is it important to demonstrate that the reporter and the protein of interest are co-expressed, but it is also important to demonstrate that the reporter does not exhibit ectopic expression, beyond endogenous expression. In situ hybridization can also be utilized to characterize the co-expression of the reporter and

Fig. 2. Identification of PCR template contamination in selected BAC clones. Uncut BAC DNA preps run on 1% agarose gel. BAC DNA is in the upper box, and contaminating PCR template in the lower box. Lane 1 - Hi Mark Ladder. Lanes 3, 4, and 6 are true positive recombinants without contaminating PCR template used to generate the

An assay was developed by Chandler and coworkers (Chandler, Chandler et al. 2007) to determine the number of copies of BAC transgene integrated into the genome. 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.

Chandler and coworkers were also able to identify multiple integration sites in BAC reporters. This was achieved by employing the BAC transgene copy number assay described above and analyzing outcrossed F2 generations for the number of transgene copies (Chandler, Chandler et al. 2007). Since the level of reporter expression is dependent on transgene copy number, it is useful to identify founders that transmit a single integration

targeted gene of interest.

linear targeting construct.

**5.2 BAC transgene copy number** 

**5.3 Integration site of BAC transgene** 

site, ensuring consistent reporter expression in future progeny.
