Fig. 1.
Human globin transgene structure in the Berkeley mouse model for sickle cell disease (SCD). Berkeley mice harbor three separate human transgenes encoding α-globin (HBA1; 1.5 kb), the extended β-like globin locus (HBBS-HBD-HBG1-HBG2; 39 kb) and a mini-locus control region (LCR; 6.5 kb), and lack endogenous mouse globin genes. (A) Target locus amplification (TLA) sequence analysis of each human transgene and flanking mouse genomic DNA was performed in a homozygous Berkeley mouse. Graph shows sequence coverage across the mouse genome. The peak signal on mouse chromosome 1 (mchr1) indicates a single integration site for all three human transgenes. (B) Metaphase fluorescence in situ hybridization (FISH) analysis of Berkeley mouse fibroblasts using probes for human HBB (red) and a control region of mchr1 (green). (C) Map of mchr1 near the transgene integration site (TIS). Human transgene configurations derived from a subset of TLA fusion reads is shown for HBA1 (pink), HBB (blue) and mini-LCR (teal) fragments with directionality indicated by arrowheads. The diagram does not represent a precise order of integrated transgenes or include all copies. (D) Copy number estimation of HBB, HBA1, HBG2 and mini-LCR by droplet digital PCR (ddPCR) of genomic DNA from Berkeley mice (n=6) heterozygous for the human transgenes, relative to the single-copy mouse gene Fzd2. (E) The top panel shows FISH images of heterozygous Berkeley mouse lung fibroblasts labeled with BAC clones flanking the transgene TIS (centromeric: RP23-308J23, red; telomeric: RP24-347O20, green). The bottom panel shows the same cell with the human transgene-containing chromosome labeled with an HBB probe (RP11-622D14, red).