Transgene copy number of the 4 transgenic lines was determined using absolute quantification-based real-time PCR (Yuan et al., 2007). PCR reactions were performed with primers designed to amplify a fragment of the β-gal coding sequence present in the β4 promoter/β-gal transgene. The sequence of the upper strand primer was 5′-GATTTCCATGTTGCCACTCGCTTTA-3′ while that of the lower strand primer was 5′-TTCAGCAGCAGCAGACCATTTTCAA-3′. All PCR reactions were set up in triplicate and included 100 nanograms of genomic DNA as template. Two positive control samples of known copy number (a generous gift from Ricardo Medina) were used in this analysis. These control samples were isolated from transgenic animals that contain a targeted β-gal coding sequence. One of these animals has 1 copy of the β-gal transgene and the other has 2 copies. The value obtained for the single copy positive control sample was set as 1 in each experiment. This value was used to estimate the copy number for all other samples including the control sample with 2 copies. In quadruplicate assays run with the 2-copy control, transgene copy numbers ranged from 2.005 to 2.274
