Figure 6: Schematic diagram for the generation of biallelic knockout mutants.

Strategy for the generation of biallelic knockouts is to replace a critical exon of one allele of the target gene with a drug selection cassette by homologous recombination and to screen clones for damage to the second allele induced by error-prone non-homologous end-joining (NHEJ). Since only one copy of the target exon will be present in correctly targeted clones, NHEJ-induced damage to the non-targeted allele can be simply assessed by Sanger sequencing of PCR products from the target exon.