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. 2021 Dec 13;82(4):615–631. doi: 10.1158/0008-5472.CAN-21-1845

Figure 1.

Figure 1. Generation of a cDNA-based complementation system for the functional analysis of human CHEK2 variants. A, Schematic representation of the mES cell– and cDNA-based complementation system for functional analysis. The DR-GFP reporter and RMCE have been stably integrated at the Pim1 and Rosa26 loci, respectively. Endogenous mouse Chek2 was targeted with CRISPR/Cas9 using a gRNA against exon 3. B, Western blot analysis of the indicated proteins from unirradiated and IR-exposed (10 Gy) Chek2WT and Chek2KO mES cells. Tubulin was used as a loading control. C, Western blot analysis of the indicated proteins from IR-exposed (10 Gy) Chek2WT, Chek2KO, and Chek2KO mES cells complemented with human CHEK2 cDNA. Tubulin was used as a loading control. D, Schematic representation of the CHK2 protein, with variant positions indicated and categorized as either synonymous (green), truncating (red), and missense VUS (blue). The amino acid numbers are shown to demarcate CHK2's evolutionarily conserved functional domains. (T) refers to the T-loop or activation segment.

Generation of a cDNA-based complementation system for the functional analysis of human CHEK2 variants. A, Schematic representation of the mES cell– and cDNA-based complementation system for functional analysis. The DR-GFP reporter and RMCE have been stably integrated at the Pim1 and Rosa26 loci, respectively. Endogenous mouse Chek2 was targeted with CRISPR/Cas9 using a gRNA against exon 3. B, Western blot analysis of the indicated proteins from unirradiated and IR-exposed (10 Gy) Chek2WT and Chek2KO mES cells. Tubulin was used as a loading control. C, Western blot analysis of the indicated proteins from IR-exposed (10 Gy) Chek2WT, Chek2KO, and Chek2KO mES cells complemented with human CHEK2 cDNA. Tubulin was used as a loading control. D, Schematic representation of the CHK2 protein, with variant positions indicated and categorized as either synonymous (green), truncating (red), and missense VUS (blue). The amino acid numbers are shown to demarcate CHK2's evolutionarily conserved functional domains. (T) refers to the T-loop or activation segment.