Identification of embryonic stem (ES) clones bearing Baf57Int and Baf57ΔR. (A) PCR screening for ES clones with correctly integrated arms. We first used the primer pair a/b (Figure 2) to screen for left-arm integration. Of twenty clones examined, seven gave positive results; five of these seven clones were confirmed by re-screening (top), and in each of the five clones, the right arm was correctly integrated based on PCR using primer pair c/d depicted in Figure 2 (bottom). (B) To determine if the 5' LoxP site is present in these five clones, we digested the PCR products amplified with the primer set a/b with restriction enzymes recognizing cloning sites at the LoxP site, which showed that clone 5 carried the 5' Lox P site and hence the Baf57Int. Of note, an additional EcoR1 site was present within the gene-trapping cassette, hence there were cleavages in all five amplicons. The 3.4 kb amplicon (Figure 2) is depicted at the bottom, with the approximate positions (in kb) of the restriction sizes indicated. The green arrows denote PCR primers as in Figure 2. C, ClaI, E, EcoR1, N, NdeI. (C) Southern blotting confirmed the identities of Baf57Int and Baf57ΔR. The asterisks and arrows indicate the genomic fragments released (by EcoRV digestion) from the WT and the targeted alleles, respectively. The strategy of the assay is diagrammed at the bottom.
Chaiyachati et al. BMC Biology 2012 10:96 doi:10.1186/1741-7007-10-96