Site-specific integration of <i>Sry</i> transgenes 3′ to the <i>Col1A1</i> gene.

<p>KH2 ES cells carry an Frt-PGK-neo-pA-Frt-hygro-pA “homing” cassette at a <i>Pst</i>I site downstream of the <i>Col1A1</i> locus. <i>Sry</i> transgene constructs were cloned into a PGK-ATG-Frt vector, and then this was integrated into the homing site within the KH2 cells by co-electroporation with a FlpE transient expression vector (pCAGGS-FlpE-puro). Flp-Frt homologous recombination resulted in loss of the PGK-neo-pA cassette and insertion of the <i>Sry</i>-PGK-ATG-Frt vector bearing the transgene into the <i>Col1A1</i> locus in the ES cells. Black arrowheads indicate Frt sites, black boxes indicate exons, grey arrows and italics indicate primer sites for genotyping of ES cells and mice. Amp, ampicillin resistance. Neo, neomycin resistance. Hygro, hygromycin resistance. pA, polyadenylation signal. PGKp, PGK promoter. Not drawn to scale.