Figure 1. The RCAN1 gene and production of BAC-Tg mice.

A, The RP11-272A3 BAC contains the complete RCAN1 gene flanked by 44 kb upstream and 42 kb downstream sequence (nucleotides 34,953,503-34,768,332 of the 2006 NCBI36/hg18 human genome assembly). There are three transcripts of RCAN1 according to Entrez Gene; the two major mRNA isoforms are diagrammed here. The HA.Flag epitope tag consisting of 17 amino acids was inserted before the stop codon of the last exon of the RCAN1 gene using BAC recombineering prior to creation of the Tg mice. B, Copy number analysis of the BAC-Tg in the three mouse lines by Southern blotting with a human RCAN1 probe and a control probe for the mouse Col6a1 gene. Mouse tail DNA and human genomic DNA was digested with EcoRV and used for Southern blotting. The human probe (841 bp long; 44% GC) recognizes a 4.8 kb fragment, while the mouse probe (550 bp long; 58% GC) recognizes a 8.9kb fragment. Wt: wild type mouse DNA; Hu: human DNA. The BAC copy number is estimated as 1, 3 and 5 for the RCAN1^BAC-Tg1, RCAN1^BAC-Tg2 and RCAN1^BAC-Tg3 lines respectively. C, Eight PCR primer pairs were used for verifying integrity of the BAC Tg in each mouse line. The position of the human RCAN1 probe used for Southern blotting is also indicated on this map (grey rectangle).