2158 Nucleic Acids Research, 2006, Vol. 34, No. 8 were included in this large transgene. A rigorous test of locus control was to determine whether these elements were able to establish a chromatin environment that was autonomous and independent of its site-of-insertion in the host genome. As background for these studies, it has been previously demon- strated that the endogenous rDBP locus is primarily expressed in the liver with 100- to 1000-fold lower levels observed in kidney, placenta/yolk sac, testis and abdominal fat and trace levels in intestine and spleen (4). Northern blot analyses of the 105kb-hDBP transgene detected high levels of human DBP mRNA in the liver of all lines, whereas signals of much lower intensity were consistently observed in the kidney ( 2–10% of liver) (Figure 2A, upper panel and summary table). Even lower levels were detected in the intestines of 3 of 5 lines ( 0.6–1.0% of liver), and sporadically in testes and spleen (Figure 2A and data not shown). The expression proﬁle of the endogenous mouse DBP gene (mDBP) was established in parallel (Figure 2A, middle panel). Overall, the expression proﬁle of the 105kb-hDBP transgene was consistent with that of the endogenous mDBP gene. These data indicated that the 105kb-hDBP transgene contains regulatory compo- nents sufﬁcient to establish native patterns of expression and that the mouse has all the regulatory factors necessary to support the native expression pattern of the human DBP gene. The ability of the 105kb-hDBP transgene to establish a fully productive and autonomous chromatin domain was further Figure 2. The 105kb-hDBP transgene programs full level, tissue-specific and copy number dependent expression. (A) Tissue specificity of 105kb-hDBP transgene expression. The autoradiograph shows a Northern blot surveying a set of tissues from a representative 105kb-hDBP transgenic line (mouse line 6). RNAs from the indicated tissues and control RNA from wild-type mouse liver (WT) were analyzed. The Northern blot was hybridized with an hDBP cDNA fragment and then with a mouse ribosomal protein L32 (mrpL32) cDNA fragment to normalize for RNA loading. The hDBP cDNA probe was specific for the human DBP mRNA and did not cross-hybridize to the mouse DBP mRNA. The blot was stripped then re-hybridized with rat DBP cDNA to detect the cross-hybridizing, endogenous mouse DBP mRNA. The 105kb-hDBP transgene was expressed predominantly in the livers and at lower level in the kidney of each line. The table summarizes the tissue distribution of transgene expression in an adult male mouse of each transgenic line (-; no signal detected). Expression in the liver is defined as 100 and all values in other tissues are calculated as using this as a reference. Very low-level expression was detected in intestines of three lines and occasionally in spleen or testis. (B) Copy number dependency of 105kb-hDBP transgene expression. Northern blots containing liver RNA from the five 105kb-hDBP transgenic lines were hybridized with hDBP cDNA and then mrpL32 cDNA. Three animals from each line were analyzed and a representative Northern blot is shown. hDBP mRNA was quantified for each line and normalized to the mrpL32 signal to correct for RNA loading. The corrected hDBP expression value was divided by transgene copy number and the mean ratio for line 1 was arbitrarily set to 1.0. The ratios from the remaining lines were normalized to line 1 and were plotted on the bar graph (±SD). All quantifications were done by phosphoimager analysis and all were in the linear range of detection (Materials and Methods). There were no statistically significant differences among the lines when hDBP expression was normalized to transgene copy number. A radial immunodiffusion assay was used to quantify levels of serum hDBP in each transgenic line. The serum hDBP levels per transgene copy number were plotted in the bottom panel using a logarithmic scale. The results demonstrated that the liver hDBP mRNA and serum hDBP levels were remarkably consistent from line to line in a copy number dependent fashion.