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. 2004 Oct 26;32(19):e143. doi: 10.1093/nar/gnh136

Figure 2.

Figure 2

Cre recombination deletes the Bsr selectable marker. (A) Genomic sequences flanking the insertion site of gene DDB0183838 depicted in Figure 1C were amplified by PCR and analyzed by agarose gel electrophoresis. WT is the endogenous wild-type gene sequence of ∼450 bp. Knockout mutants (KO) are clonal isolates of cells transformed and selected for resistance to Blasticidin S; insertion of the floxed-Bsr cassette yields a fragment of ∼2 kb. (B) A KO isolate from Figure 2A was transformed for transient expression of Cre, and several clonal isolates were randomly screened for functional recombination by PCR as in Figure 1A. Wild-type is the endogenous WT gene sequence of ∼450 bp. KO is the gene sequence carrying the insertion of the floxed-Bsr cassette (∼2 kb). Recombination between loxP sites generates a fragment that is ∼520 bp. The fragment was sequenced for confirmation (see Figure 1D). (C) Genomic DNA from WT, KO and Cre cells were probed by southern blot hybridization for the presence of Bsr and Neo gene sequences. DNA from a Neo-expressing transgenic cell line was included as a control.