2156 Nucleic Acids Research, 2006, Vol. 34, No. 8 Figure 1. Generation of transgenic mouse lines carrying the 105kb-hDBP transgene. (A) Isolation and characterization of the 105kb-hDBP transgene. The arrangement of the human DBP, ALB, AFP and AFM genes and their transcriptional orientations (bent arrows) are shown at the top. Clones were isolated from a human PAC library by PCR with a series of hDBP probes and the insert ends were sequenced using T7 and SP6 primers (25). PAC clone 231M2 encompasses a 105 kb human genomic fragment containing the entire 42kb-hDBP gene with extended 50- and 30-flanking regions. Representative partial digestion patterns of 231M2 using four restriction enzymes indirectly end-labeled with a T7 probe are shown in the autoradiograph to the right. The deduced restriction map of 231M2 and the position of hDBP within the 231M2 fragment derived from this analysis are summarized at the left. (B) Mapping and copy number determination in five independent 105kb-hDBP transgenic lines. The 105kb-hDBP transgene was released from PAC clone 231M2 by digestion with NotI (A), purified, and microinjected to generate transgenic founders. A series of five transgenic mouse lines were obtained. The transgene copy numbers were determined in the F1 generation of each line by Southern blot analysis. Tail genomic DNA was digested with SacI and hybridized with an hDBP cDNA probe (303 bp EcoRI fragment). Genomic DNA from HeLa cells and the tail of a wild-type mouse were used as positive and negative controls, respectively. The lower diagram shows the 4.0 kb SacI fragment expected to hybridize to the probe, as well as the location of this cDNA probe. The transgene-specific bands (4.0 kb, indicated by arrow) were quantified by phosphorimaging. Transgene copy numbers were estimated by normalizing these signals to those from human genomic DNA (HeLa cell; two endogenous copies). The mouse z-globin was used as the DNA loading control. The deduced transgene copy numbers in the five lines ranged from 2 to 12 and are indicated below the corresponding lanes. fragment. The transgene copy number for each line was determined by Southern blot analysis. All animal work was carried out under protocols approved by the University of Pennsylvania Institutional Animal Care and Usage Committee. Southern blot analysis An aliquot of 10–15 mg of restriction enzyme-digested mouse tail DNA was analyzed on 0.8% agarose gels, transferred to Zetabind nylon membrane with 10· SSC (1.5 M NaCl, 0.15 M sodium citrate), ultraviolet (UV) cross-linked to the mem- brane. The membrane was prehybridized [0.5 M NaPO4 (pH 7.2), 7% SDS, 1% BSA and 200 mg/ml denatured salmon sperm DNA] at 65 C, hybridized at 65 C with 32P-labeled probe for 16 h, subsequently washed in 0.5· SSC, 0.1% SDS at room temperature, and ﬁnally 0.1· SSC, 0.1% SDS at 65 C. The washed membranes were exposed to XAR-5 ﬁlms (Kodak), and signals were quantiﬁed by phosphorimager (Molecular Dynamics, Inc., Sunnyvale, CA).