Humanized chromosomes used to study effect of ectopic reinsertion of HS −40. (A) An outline of the 3 recombinant humanized chromosomes analyzed in this study: (top) humanized chromosome with intact normal sequence in which HS −40 is located within the C16orf35 gene (referred to as normal humanized chromosome); (middle) humanized chromosome with HS −40 deleted from the C16orf35 gene (referred to as ΔHS −40 chromosome); (bottom) humanized chromosome with HS −40 deleted from the C16orf35 gene and relocated to an ectopic position (referred to as 3′HS −40 chromosome). Human genes are represented by gray filled rectangles; mouse genes, by open rectangles. Human and mouse sequences have boundaries in Dist and theta genes indicated by m/h and h/m. Human/mouse boundaries and positions of deletion and reinsertion of HS −40 correspond to the position of site-specific recombination sites (key) used to engineer these rearrangements. For a more detailed explanation of the chromosome engineering steps involved, see supplemental Figure 1A and B. (B) Southern blot analyses of the HS −40 reinsertion site with a PstI α probe that hybridizes with the α-globin genes. Genomic DNA was digested with BglII and HindIII and run on a 0.65% agarose gel. From left to right: genomic DNA from mouse, human, humanized mice lacking HS −40, normal humanized mouse, and with the reinsertion of HS −40 at the 3′ end of the cluster. Arrowheads show a size increase (1.1 kb) in the genomic DNA fragment of mice with the reinsertion of HS −40 between the α-globin gene and the θ gene. The normal 9.4 kb (open arrowhead) BglII fragment is increased to 10.5 kb (closed arrowhead) and the normal 4.1 kb (open arrowhead) HindIII fragment is increased to 5.2 kb (closed arrowhead). A small restriction map is represented at the bottom of panel A.