Table 1. Oligonucleotide primers and probes Probe P1 P2 P3 P4 P5 P6 pI pIII Primer sequence 50-TGGGGCACAATGGAATAGTTTG-30, 50-CCGAGATGCTGAAACAACCTAAGTG-30 50-AGAAGACTGTCGCAGCATTAAAGG-30, 50-CAGATGGGGAAAAAATGGGTG-30 50-CGCCTGAGTCTTTCAACCAATCTG-30, 50-CCCTGCATTTAATTTGGACGC-30 50-CATCTGGGAAAGGAGGACTTCAC-30, 50-CTTAGGGCTTCCATCAAACCAATC-30 50-ACTCCAACCTGGGTGACAGAGG-30, 50-GCTTTGCGAAACAATGAGGATG-30 50-GCTTGAGAGTTTACCCAATTTCCA-30, 50-ACCTGATTATCAATGACTGAGATC-30 50-GAATCACAGTAAGCAATCAA-30, 50-CTTTATCTCTGCTTTTGCAA-30 50-TCCCACTAAACAGATCTCAGTCAT-30, 50-GCAAGGACTTCATGTCTGAAACAG-30 Northern blot analysis Total RNA was extracted from various tissues with RNAzol. 5–20 mg of total RNA were denatured at 55 C, separated in 1.5% agarose-formaldehyde gels, and transferred to Zetabind membrane with 10· SSC. After UV cross-linking, the blots were prehybridized, and subsequently hybridized with 32P-labeled probes at 42 C for 16 h. The membranes were washed with 2· 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). Radial immunodiffusion assay One percent agarose containing 3% rabbit polyclonal anti-hDBP (Cocalico Biologicals Inc., Reamstown, PA) was poured on to a glass backing, and circular wells were cut into the solidiﬁed matrix. Test mouse sera and the standard sera containing hDBP protein (Calbiochem Inc., San Diego, CA) (2–4 ml), were loaded into each well and allowed to diffuse for 40 h at room temperature. The gels were rinsed ﬁrst with phosphate-buffered saline (PBS) for 16 h, then with distilled water for 20 h. Gels were stained with 0.1% Coomassie brilliant blue in 50% methanol and 10% acetic acid for 30 min, and subsequently de-stained with 50% methanol and 10% acetic acid for 1 h. The amount of hDBP in each serum sample was obtained by comparing the diameters of the stained immunodiffused circles of each test serum and the hDBP standard sera (50–500 mg/ml). Isolation of intact nuclei Livers of 105kb-hDBP transgenic mice were perfused with cold PBS and minced. Liver nuclei were isolated as described (29). The nuclei pellet was resuspended in buffer D [15 mM Tris–HCl (pH 7.4), 15 mM NaCl, 60 mM KCl, 0.5 mM EGTA, 0.5 mM b-mercaptoethanol, 0.5 mM spermidine and 0.5 mM spermine]. Brains of 105kb-hDBP transgenic mice were washed in PBS. Brain cells were dissociated in cell-free dissociation buffer (GIBCO-BRL, Grand Island, NY). Cells were lysed in NB3 buffer [320 mM sucrose, 1 mM MgCl2, 0.05% Triton X-100, 1 mM Pipes (pH 6.4) and 0.1 mM Nucleic Acids Research, 2006, Vol. 34, No. 8 2157 phenylmethylsulfonyl ﬂuoride (PMSF)]. Nuclei were washed in RB buffer [0.1 M NaCl, 50 mM Tris–HCl (pH 8.0), 3 mM MgCl2, 0.1 mM PMSF and 5 mM sodium butyrate], pelleted and resuspended in RB buffer. DNase I hypersensitivity mapping The concentrations of nuclei were estimated from measure- ments of A260/A280. 500 mg of liver nuclei were suspended in buffer D with 5 mM MgCl2 and were incubated on ice for 10 min with increasing amounts of DNase I (GIBCO-BRL). EDTA was added to 25–50 mM ﬁnal concentration to termi- nate the reactions. The DNase I digested liver nuclei were incubated in lysis buffer (800 mM NaCl, 0.5% SDS and 100 mg/ml proteinase K) at 55 C overnight. 100 mg of brain nuclei were suspended in RB buffer with 1 mM CaCl2 and incubated on ice for 10 min with increasing amounts of DNase I (GIBCO-BRL). After terminating the reaction by adding EDTA to 25–50 mM and an equal volume of lysis buffer (1.6 M NaCl, 1% SDS and 200 mg/ml proteinase K), DNase I digested brain nuclei were incubated at 55 C over- night. The lysed liver or brain nuclei samples were extracted with phenol and chloroform, and the DNAs were precipitated with ethanol and suspended in TE buffer. The DNAs were subsequently digested with appropriate restriction enzymes, resolved by electrophoresis on 0.8–1.0% agarose gels, and transferred to Zetabind nylon membranes for Southern blot analysis. RESULTS Generation of mouse lines carrying the hDBP locus with extensive flanking sequences (105kb-hDBP) In order to assess the ability of the isolated hDBP gene to establish an autonomous hepatic chromatin environment capable of supporting consistent and robust expression, a set of transgenic mouse lines were generated. To optimize the possibility of including all necessary regulatory elements, a large genomic fragment was used as transgene. The hDBP gene encompasses 13 exons and spans 42 kb (13). Four human genomic PAC clones containing the entire hDBP gene were isolated from a PAC-recombinant library (25). Among them, PAC clone 231M2 carried a 105 kb human genomic fragment encompassing the entire hDBP gene along with 37 kb of 50-ﬂanking region and 26 kb of 30-ﬂanking region (105kb-hDBP transgene; Figure 1A). This insert was released from the PAC vector, microinjected into fertilized mouse oocytes, and ﬁve founders were obtained. Each founder was crossed with a CD1 mate to generate F1 transgenic mice and ﬁve 105kb-hDBP lines were established. The integrity of the transgene in each line was determined by extensive Southern blot analysis of F1 or F2 tail DNA (Figure 1B and data not shown). Transgene copy numbers ranged from 2 to 12 as determined by Southern blot after the transgene signal was normalized to the hDBP signal in total human (HeLa) DNA (two copies) and to the endogenous mouse z-globin signal as a loading control. Expression of the 105kb-hDBP transgene The expression of the 105kb-hDBP transgene was assessed to determine whether the full complement of regulatory elements