2162 Nucleic Acids Research, 2006, Vol. 34, No. 8 Figure 5. Comparisons between human and rodent genomic sequences in the vicinity of the DBP locus. A map of the hDBP gene is displayed in the center of the figure showing the exon-intron arrangement and location of the HS in liver (downward arrows) and brain (upward arrows). An expanded view of the sequence comparisons (human compared to rodent) in the 50-flanking region (upper panel) and intron 1 (lower panel) are shown. The numbering on the x-axis refers to the position in the human sequence. The level of similarity in sequence is displayed on the y-axis as a percentage (mVISTA program; window size 50 bp, homology threshold 70%). The 75% level of sequence similarity is marked by a horizontal line and regions of the sequence representing repeated elements are indicated by the shaded rectangles, LINE elements (stippled) and SINE (solid). The positions of liver-specific HSI, HSIII, HSIV and HSV sequence comparisons are each indicated by the downward arrows. Note the peaks of sequence identity at each of the indicated DNase I HS. Figure 6. Generation of transgenic mouse lines using the 51kb-hDBP transgene. (A) Map of the 51kb-hDBP transgene. PAC clone 45P24 (25) was digested with NotI and FspI to generate the 51kb-hDBP transgene. This 51 kb transgene encompasses the hDBP gene with 2.5 kb of 50-flanking and 6.5 kb 30-flanking regions, including all of the liver-specific DNase I HS detected. Positions of probes used to confirm the structure of the transgene are indicated (pI, pII, derived from the hDBP cDNA, and pIII; Table 1) (B) Southern blot analysis of PAC clone 45P24 confirmed the structure of the 51 kb fragment. Insert DNA was released with NotI digestion from the PAC vector and the 51 kb fragment was obtained with NotI and FspI double-digestions. Southern blots of these digestions were probed with radiolabeled pI, pII or pIII (A). A single 51 kb band was detected with each probe as expected. (C) Analysis of 51kb-hDBP transgenic lines and copy number determinations. Four 51kb-hDBP mouse lines were generated (910D, 924B, 955B and 955E). The transgene copy numbers were determined in F1 or F2 generations of each line by SacI digestion and Southern blot analysis as described (Figure 1B). The copy numbers determined (ranging from 6 to 22) are indicated below the corresponding lines. sequences and 26 kb of 30-ﬂanking sequences (105kb-hDBP transgene) paralleled that of the endogenous DBP locus. This result represents the analysis of ﬁve independent lines. Not only was the robust expression primarily restricted to the liver, but the major secondary site of DBP expression in the kidney was also preserved (Figure 2A). Of particular note, the level of transgene expression, when monitored at the protein level and normalized to transgene copy number, was remarkably