The human JAK2-V617F (Flip-Flop) transgene. (A) Transgenic construct. A human BAC containing JAK2 exons 1-12 was combined with a partial cDNA encoding JAK2 exons 13-25 (black box; not to scale) and a polyadenylation signal (white box). (B) Inducible transgenic construct. The cDNA is placed in the inverse orientation (white arrow) and is flanked with mutant loxP sites (magnified insert). Cre-mediated recombination flips the orientation of the cDNA and places the intron 12 splice acceptor (SA) into the correct position to allow proper splicing of the transgenic pre-mRNA. Recombination generates one wild-type loxP and one double mutant lox66/77 site, which is no longer substrate for the Cre-recombinase. The position of the V617F mutation is indicated by a red arrow. (C) Localization of the transgene in the transgenic strain FF1. Fluorescent in situ hybridization (FISH) shows that the transgene integrated into a single locus, which appears as 2 red signals in metaphase chromosomes (red arrow in left panel). Spectral karyotyping (SKY) identified chromosome 8, band A1 as the transgene integration site (red arrow in right panel). (D) Model of Cre-mediated rearrangements in the transgenic integration site. A perfect head-to-tail orientation of all transgenic copies is assumed. In the FF1 strain we found that 9 copies of the transgene have integrated, of which only 3 are shown. The native configuration, in which all transgenic copies are in the inactive orientation (red boxes), is shown on top. Cre-recombination between adjacent loxP sites (green arrows) leads to reversal of the orientation and activation of the transgene (green boxes). A maximum of 9 active transgene copies can be generated (middle panel). Cre-recombination between distant loxP sites that are in a parallel orientation (red arrows) results in the excision of one ore more copies of the transgene (bottom panel).