Fig. 1

Development of a cDNA-based complementation system for the functional analysis of human PALB2. a Schematic of the cDNA-based complementation system for functional analysis of human PALB2. The DR-GFP reporter for HR and recombination-mediated cassette exchange system (RMCE) for site-specific integration and expression of a human PALB2 cDNA were incorporated at the mouse Pim1 and Rosa26 (R26) loci, respectively. Endogenous mouse Trp53 was targeted with CRISPR/Cas9 using a gRNA against exon 1, whereas endogenous Palb2 was targeted with a gRNA against exon 4 (left). Transient expression of the I-SceI endonuclease in Trp53^KO/Palb2^KO cells expressing human PALB2 cDNA (with or without a variant) allows for assessment of the HR efficiency using the DR-GFP reporter (right). b DR-GFP assay in Trp53^KO mES cell clones transfected with an I-SceI and mCherry co-expression vector. GFP expression was monitored by Fluorescence-Activated Cell Sorting (FACS). Data represent mean percentages (±SEM) of GFP-positive cells among the mCherry-positive cells relative to that for the wild type (WT), which was set to 100%, from two independent experiments (left). Western blot analysis of Trp53 expression in 4 Trp53^KO mES cell clones. Histone 3 (H3) was a loading control (right). c Karyotyping of Trp53^KO mES clones from (b). The bar graph shows the percentages of cells with 40 chromosomes (n = 50 cells per condition). d DR-GFP assay in Trp53^KO and Trp53^KO/Palb2^KO mES cells expressing WT PALB2 or not. Cells were transfected with an I-SceI and mCherry co-expression vector. GFP expression was monitored by FACS. Data represent mean percentages (±SEM) of GFP-positive cells among the mCherry-positive cells relative to that for Trp53^KO cells, which was set to 100%, from four independent experiments (left). Western blot analysis of mouse (m)Palb2 expression in Trp53^KO and Trp53^KO/Palb2^KO (clone 3) mES cells (right). An unspecific band was a loading control (right). Source data are provided as a Source Data file.