A floxed GFP/hygromycin resistance cassette was placed upstream of exon 3 and an additional loxP site was placed downstream of exon 4. Chimeric mice were bred with Tg(rtetR-TetO-cre)40Mhz mice to remove the selection cassette and exons 3 and 4. Gene inactivation was confirmed by a lack of protein detected in immunoblot analysis of E9.5 embryo lysates.