Homologous recombination (HR) in founder mice.

<p><b>(a)</b> Genomic DNA was amplified with primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.t004" target="_blank">Table 4</a>) designed to flank the <i>Tyr</i> mutation site resulting in a PCR product of 1555bp (solid arrow). Amplification of mice 3.1 and 4.9 yielded multiple smaller molecular weight bands. The bands represented by asterisks have been confirmed by sequence analysis and represent an alternate truncated allele. Interestingly, the phenomenon of heteroduplex formation (arrowheads) is also evident in mice with more than one allele size. These artifacts are thought to occur upon heterologous binding of DNA strand from each allele (Thompson et al. 2002). Sanger sequencing of predicted heteroduplex bands confirmed the mixture of both higher and lower molecular weight bands in the product. To eliminate interference with the restriction analysis, the 1555bp band was excised from the gel for all of these mice and subjected to <i>Sal</i>I digest <b>(b)</b>. <b>(c)</b> Black-eyed progeny from mouse 4.9 all appeared to be positive for donor insertion. The predicted digestion products of 835bp and 720bp are indicated by the dashed arrow. The 100bp molecular weight marker was loaded in the left-most lane of each agarose gel with sizes as indicated (in Kb). B6: C57BL/6, AB6: C57BL/6 Albino. Mouse numbers are consistent with the text.