2160 Nucleic Acids Research, 2006, Vol. 34, No. 8 Figure 3. Two liver-specific DNase I HS are present 50 to the hDBP gene. (A) Mapping the 50-flanking DNase I HS using a downstream probe. A BglI (B) restriction map of the hDBP gene and flanking regions from position 37 to +68 kb is shown at the top. The position of hybridization probe P1 is labeled and the 26 kb BglI fragment subjected to mapping is indicated (horizontal double-headed arrow). DNase I HS mapping was carried out on chromatin isolated from 105kb-hDBP liver and brain (negative control), left and right autoradiographs, respectively. Aliquots of chromatin were subjected to increasing amounts of DNase I (wedges). Genomic DNA from these aliquots were digested to completion with BglI and subjected to Southern analysis. The 26 kb BglI fragment spanning the 50-flanking region, exon 1 and part of intron 1 was mapped by hybridization to probe P1 (see Table 1). Two prominent liver-specific DNase I HS (HSI and HSIII) were identified near the hDBP- proximal promoter region. A single weak HS (HSII) was identified only in the control brain sample. The results are summarized on the map under the autoradiographs. Vertical arrows indicate the positions of the DNase I HS (#; liver, "; brain). The horizontal arrows below the map indicate fragment sizes (kb) and their designations. (B) Refined mapping of the 50-flanking DNase I HS using an upstream probe. The DNase I mapping results in (A) were confirmed using a probe located upstream of the sites and at a higher level of resolution. The map at the top shows the 6.7 kb HindIII (H) fragment that encompasses the identified DNase I sites and the location of probe P2 (Table 1). The Southern analysis of liver chromatin samples as in (A) confirmed the presence of the two liver-specific DNase I HS (HSI and HSIII). In the analysis of this shorter fragment, the brain-specific site was resolved into two closely linked HS (designated HSII-1 and HSII-2). The results of the refined mapping are summarized on the map under the autoradiograph, as in (A). northern blot analysis at trace levels in the kidneys of two of the four 51kb-hDBP lines (0.05 and 0.04% of expression levels in livers, respectively); in the remaining two 51kb- hDBP lines expression in the kidney could not be detected (Figure 7A table and data not shown). Serum hDBP levels in the four 51kb-hDBP lines varied by less than 3-fold (80–189 mg/ml/transgene copy), indicating copy number dependent expression (Figure 7B). These levels were remark- ably similar to those observed in the sera of 105kb-hDBP transgenic mice and similarly approximate the levels of expression of the native hDBP gene (1) (Figure 2B). These data demonstrated that the 51kb-hDBP transgene, containing only 2.5 kb of 50-ﬂanking region and 6.5 kb of 30-ﬂanking region, was able to overcome site-of-integration position effects and direct high levels of liver-speciﬁc hDBP gene expression from multiple random integration sites in trans- genic mouse chromatin. In addition, we note a selective loss of hDBP transgene expression in the kidneys of the 51kb-hDBP mice when compared with the 105kb-hDBP trans- gene, suggesting that expression in the kidney may rely on a distinct set of determinants. DISCUSSION In the present report we have tested for autonomous regulation of the hDBP gene and have sought sites in the native hepatic chromatin that might encompass relevant regulatory elements. Many transgenes are not consistently or accurately expressed in the mouse due to the exclusion of determinants that are necessary to re-establish the native chromatin environment. Thus these transgenes are sensitive to the major inﬂuences, both positive and negative, at the foreign site-of-integration in the host (mouse) genome. An LCR is deﬁned by its ability to re-establish a chromatin environment that is dominant over site-of-integration effects. Thus the LCR action allows the transgene to be expressed at a consistent level when compared among multiple lines, each representing a unique insertion site. The initial approach in our study was to introduce the hDBP gene with extensive ﬂanking sequences into the mouse genome and determine whether it could establish an appropri- ate pattern and level of gene expression. The results clearly demonstrated that this was the case. The pattern of expression of the hDBP transgene encompassing 37 kb of 50-ﬂanking