Fig. 3

Homozygous mice were identified using traditional PCR of genomic DNA. Oligonucleotide primers designed to amplify parts of the TTR-tdTomato BAC were combined in PCR reactions with primers to amplify control sequences present in all mice. These reactions were subjected to conventional 1% agarose gel electrophoresis (a). Images of the gels were then analyzed as densitometry scans using Image J, and the relative areas under the ttr-BAC amplicon band (t) and the control amplicon band (c) were calculated as a ratio (b). The ratios obtained for any one BAC sequence were plotted against the ratios for each other BAC sequence (c). Certain mice from a given litter tended to have high ratios of BAC to control amplicons across all primer pairs, yielding distinct clusters in these plots (e.g., mice 891 and 892 in c). Nine out of ten mice identified this way, including 891 and 892, were verified to be homozygous by breeding with wild-type mates. The differences in ratios typically reflected both an increase in the fluorescence intensity of the BAC amplicon and a decrease in intensity of the control amplicon for the homozygous mice (b), suggesting competition between these reactions