Fig. 4. Repair of DR1Bsd leads to blasticidin resistance by homology-directed repair. (A) Bar graph showing the number of blasticidin-resistant colonies per thousand cells plated (corrected for transfection and cloning efficiencies). The left column represents Brca2Tr/Wt cells containing S1Bsd alone and the right column Brca2Tr/Ex27+ cells that contain both the S1Bsd and the homologous donor repeat 5′ΔBsd in DR1Bsd. There is very little induction of blasticidin resistance in S1Bsd; therefore, the frequency of repair of a DSB in S1Bsd relative to wild-type Bsd by NHEJ is extremely low. Error bars represent ± 1 SEM. (B) A representative Southern blot of BglII–SalI-digested genomic DNA from Brca2Tr/Ex27+ or Brca2Tr/ΔEx27 ES cells before (marked No Tx) and after I-SceI-induced DSB repair and subsequent blasticidin selection (marked Bsdr). A TK promoter fragment (indicated in Figure 3) was used as a probe. A dominant 2.8 kb restriction fragment is seen in both cell lines before transfection of pCAGGS 3 × nls I-SceI and is from the unbroken DR1Bsd substrate. After DSB induction, repair and selection of blasticidin-resistant colonies, the predicted 0.84 kb HR fragment is dominant in all cell lines. This arises due to HR with 5′ΔBsd and transfer of the wild-type SalI-containing sequence from 5′ΔBsd to S1Bsd, to create Bsd.