Fig. 3.

Confirmation of pop-in and pop-out genotypes. (a) Successful pop-in gene targeting produces a tandem integrant with one wild-type and one modified version of the target locus. These are readily identified by PCR with one primer outside of the region of homology used for targeting and one primer within non-homologous portions of the targeting vector (top, primer pairs 1–2, 3–4, or both). In rare cases, the locus can have two wild-type or two modified alleles (see text for details). These can be identified by the presence or absence of an RFLP in the diagnostic PCR products (top) or by RFLP analysis of PCR products amplified, simultaneously in one reaction, from both copies using primers inside the region of homology (bottom, primer pair 5–6). (b) Pop-out of the targeting vector leaves a single copy of the locus, which is confirmed by PCR using primers located outside the region of homology used for targeting (primer pair 1–4). The single copy can be either wild-type or modified and these can be distinguished by the presence or absence of the diagnostic RFLP.