evaluated by determining the relationship between hDBP expression levels and 105kb-hDBP transgene copy number (Figure 2B). If the transgene contains locus control determi- nants sufﬁcient to establish an autonomous chromatin domain, expression of the transgene at each independent insertion site should be directly related to transgene copy number. This was tested in the case of the hDBP transgene at both the levels of mRNA expression and protein production. hDBP mRNA levels in the livers of mice from each of the ﬁve 105kb- hDBP lines were quantiﬁed by northern analyses. Levels of hDBP mRNA were normalized to endogenous mouse ribosomal protein L32 (mrpL32) mRNA as a loading control and divided by transgene copy numbers to generate an expres- sion per transgene copy value (Figure 2B, autoradiograph). A tight correlation was observed among values from the ﬁve lines, lying within a 2-fold range. The correlation between transgene copy number and levels of gene expression was conﬁrmed by analyses of serum hDBP protein levels. The serum hDBP levels/transgene copy number in adult mice from the ﬁve lines fell within a 3-fold range, ranging from 85–233 mg/ml/copy. Remarkably, the levels of serum hDBP in these 105kb-hDBP mouse lines, when corrected for transgene copy number, approximated the range of normal human serum DBP levels (116–232 mg/ml/copy) (1) (Figure 2B, graph). There was no evidence for sexual dimorphism of transgene expression (data not shown). These results demonstrated site- of-integration independent, copy number dependent and tissue-speciﬁc expression of the 105kb-hDBP transgene. This was consistent with the establishment of an autonomous chromatin domain by the transgene and furthermore was con- sistent with the presence of determinants that constitute a LCR (19,30,31). Liver-specific DNase I hypersensitive sites (HS) are established at the 105kb-hDBP chromatin locus The transgene expression studies (Figures 1 and 2) indicated that determinants sufﬁcient for establishment of a fully pro- ductive hDBP locus in native hepatic chromatin are encom- passed within the 105kb-hDBP transgene. To identify candidate regions that contain the relevant LCR determinants, DNase I mapping was performed on the 105kb-hDBP trans- genic liver chromatin. Regulatory sequences involved in such LCR function commonly localize to DNase I HS within the chromatin of expressing tissues [reviewed in ref. (32,33)]. With this tissue speciﬁcity in mind, chromatin from the brain (negative control) of 105kb-hDBP transgenic mice was also mapped with DNase I. A mapping approach was designed to cover the entire gene and its adjacent ﬂanking regions. A representative analysis of the 26 kb BglI fragment that spans the 50 terminus of the gene (from 17.4 to +8.6 kb) is shown (Figure 3A). A probe hybridizing to the 30 terminus of this fragment (probe P1) revealed two strong liver-speciﬁc DNase I HS and one weak brain-speciﬁc HS in the promoter region (Figure 3A). Higher resolution analyses of these HS were obtained by probing a 6.7 kb HindIII fragment spanning sequences 3.0 to +3.7 kb (probe P2) (Figure 3B). This study conﬁrmed the presence of the two liver-speciﬁc HS (HSI at 2.1 kb and HSIII at 0.13 kb) as well as the two additional HS speciﬁc to the brain (HSII-1 at 1.8 kb and HSII-2 at 1.5 kb). Nucleic Acids Research, 2006, Vol. 34, No. 8 2159 DNase I mapping was extended throughout the hDBP gene and its 30-ﬂanking region (Figure 4 and data not shown). These analyses detected four additional HS in liver: HSIV, HSV, HSVI and HSVII. HSIV and HSV are located within intron 1 (Figure 4A and B). The broader and stronger HSIV was com- posed of at least three sub-signals, located at map coordinates +10.3 kb to +10.9 kb. A weaker HSV was located at +12.2 kb. Two additional HS were observed at the 30 terminus of the gene; HSVI (+42.7 kb) coincident with the polyA site was present in both liver and brain and hence was not liver-speciﬁc. In contrast HSVII (+43.9 kb), located immediately 30 to HSVI, was liver-speciﬁc (Figure 4C). The full HS mapping strategy and a summary of the results are shown (Figure 4D). HSI is located 2.1 kb relative to the site of hDBP tran- scriptional initiation. It is of note that the sequences corre- sponding to HSI as well as HSIII are highly conserved. Comparison of the sequences at HSI demonstrated over 80% identity between human and rodent [GenBank accession no. L10641, L10642 (human), MW_047424 (rat) and NT_039308 (mouse)], (Figure 5, upper). Such conservation of non-coding sequences in genomic alignments is a reliable indicator of regulatory determinants (34–36). The proximity of HSI with HSIII suggested that the two might work in concert to coordinate hDBP promoter function. Genomic sequence align- ments also revealed a remarkable conservation of non-coding sequences within intron 1. This conserved segment is coinci- dent with the 1.9 kb region bracketed by HSIV and HSV (Figure 5, lower). Sequence comparisons of the human and rodent DBP loci demonstrated that HSVI and HSVII in the 30 terminus region also correspond to segments with over 70% sequence similarity (data not shown). Thus, a set of liver- speciﬁc HS was identiﬁed at the 105kb-hDBP transgene locus in hepatic chromatin and these sites are coincident with segments of conserved non-coding sequences in the 50-ﬂanking region, in intron 1, and in the 30-ﬂanking region. This concordance of HS mapping and localized segments of phylogenetically conserved non-coding sequences supports functional roles for these regions in hDBP expression. Generation of 51kb-hDBP transgenic mouse lines Determinants involved in chromatin organization and LCR activity are characteristically located quite distant from a tar- get gene. The HS identiﬁed in the preceding studies (Figures 3 and 4) are in close proximity to, or within, the hDBP tran- scription unit. To test whether these gene-proximal HS are sufﬁcient for hDBP activation, a second set of mouse lines was generated using a transgene with minimal ﬂanking sequences. This 51 kb genomic fragment, excised with NotI and FspI from the 123 kb human genomic PAC clone (PAC 45P24), encompassed the entire hDBP gene along with only 2.5 kb of 50-ﬂanking and 6.5 kb of 30-ﬂanking region (51kb- hDBP transgene; Figure 6A and B). Four transgenic founders were generated. Transgene integrity and copy numbers were determined for each line (Figure 6C). Liver-specific and copy number dependent expression of the 51kb-hDBP transgene High-level hDBP mRNA expression was detected in the livers of all four 51kb-hDBP transgenic lines (Figure 7A autoradio- graph and data not shown). hDBP mRNA was also detected by