Figure 5. Generation and characterization of isogenic Xlf^−/−Paxx^Δ/Δ pre-B cell lines.

(a) Scheme for murine Paxx locus and CRISPR/Cas9 mediated knockout strategy. The open triangles indicate the location of the guided RNA. The horizontal arrows indicate the location of the primers used for primary screen. Southern blot confirmation was performed using BamHI-digested DNA with the mPaxx probe (show as thick black line). (b,c) Southern blot and western blot assays confirmed the homozygous deletion of Paxx and the absence of Paxx protein in WT or Xlf^−/− v-abl transformed pre-B cells with inverted substrates. (d) IR sensitivity of WT, Paxx^−/−, Xlf^−/−, Xlf^−/− Paxx^Δ/Δ and Xrcc4^−/− pre-B cells measured, determined by cell counting. At least triplicates were plated and counted for each genotype at each dosage. (e) Left: frequency of metaphases with cytogenetic abnormalities of WT, Xlf^−/− or Xlf^−/−Paxx^Δ/Δ v-abl transformed pre-B cells. At least three independent experiments were performed. The data represent the average and the s.d. of the three repeats. Right: the frequency of chromatid (grey box) and chromosomal (white box) breaks measured by T-FISH analyses in the pre-B cells. The raw data were summarized in Supplementary Fig. 3E. X^−/−=Xlf^−/−, P^Δ/Δ=Paxx^Δ/Δ and X^−/−P^Δ/Δ=Xlf^−/−Paxx^Δ/Δ.