A floxed neo cassette was inserted 108 bp upstream of the E2f3a start codon, the mouse E2f1 open reading frame including the termination codon replaced the exon 1a open reading frame, and a third loxP site was inserted approximately 300 bp downstream of exon 1a. Western blot analysis in double homozygous MEFs lacking E2f1 wild-type alleles confirmed the expression of E2F1 protein.