The RPCI21 mouse PAC library (MRC Genomic Resource Center, England) was screened using a random hexamer labeled probe to Bptf exon 2. Blotting was performed in hybridization bags using 0.25 M sodium phosphate pH 7.2, 1 mM EDTA, 7% SDS, at 65°C overnight with rocking. Blots were washed 5 times for 10 min with 0.25× SSC, 0.1% SDS at 65°C. Eight positive clones were identified using X-ray film as; 367-I21, 373-D5, 402-C16, 402-E15, 426-P15, 540-B9, 625-D23, 582-P16. Clone 367-I21 was confirmed by Southern blotting using Eco RI and Sal I digests and Bptf exon2 probe. Genomic sequence for the construction of the targeting vector was retrieved into bluescript SKII (Stratagene) and the integration of loxP sites and Neo selectable marker was performed using recombineering technology described previously [65]. The sequence of the targeting vector is available upon request.
