A loxP site and puro cassette were inserted upstream of exon 3 and a loxP site was inserted downstream of exon 3 via homologous recombination. Cre mediated recombination removed the puro cassette and exon 3. Semi-quantitative and real-time RT-PCR analysis confirmed the absence of mRNA expression in MEFs from homozygous embryos. (J:188556)