Exon 1 was replaced with a modified exon 1 lacking the translation initation site. An FRT-flanked neo cassette was inserted upstream of exon 2. An EGFP gene with a splice acceptor and splice donor replaced part of exon 3 with additional translation reinitiation sites. Flp-mediated recombination removed the neo cassette. Western blot analysis confirmed the absence of protein expression in the forebrain, cerebellum, spinal cord and muscle and presence of Gdf1 protein expression in E10 embryos. No GFP expression is detected by western blot analysis.