Fig. 1. Targeted expression of human SPRY2 and EGFP in the ureteric bud in vivo. (A) Schematic structure of the expression construct generated by inserting a PCR fragment of human SPRY2 cDNA into the EcoRI and BamHI sites of a plasmid containing IRES-EGFP. The fragment was excised with EcoRI and SspI, and inserted into the EcoRI and SmaI sites of a modified bluescript vector. A ß-globin splice acceptor was then cut with BamHI and EcoRV, blunted and inserted into a blunted NotI site (N) downstream of the Pax2 promoter. The fragment containing the Pax2 promotor and ß-globin was removed from the vector with EcoRI and ligated upstream of the human SPRY2 cDNA, IRES EGFP-PA. The yellow arrow indicates the start site of transcription. B, BamHI; EcoRI site, (N) defective NotI site; E, EcoRI. (B) An expected 300 bp fragment was amplified with P1 and P2 primers in PCR, indicating the presence of the transgene in the genome of the carriers. (C) The copy number of the transgene was estimated by Southern blotting using GFP as a probe. Control of loading is indicated below. The Pax2 promoter drives expression of EGFP in the ureteric bud of a kidney at E11.5 (D) and E17.5 (F). The human SPRY2 gene is also expressed in the kidney at E12.5 (E) and E17.5 (G), as judged by in situ hybridization. WT, wild type; TG, transgene carrier. Scale bars: 100 µm.