Figure 1

A schematic diagram of the mammalian proten-protein interaction trap, MAPPIT. The selection system makes use of a particular HEK293 cell clone that has a Flp recombination target (FRT) integration cassette in a transcriptionally active locus. These cells also stably express the murine ecotropic retroviral receptor (mEcoR) and have a stably integrated puromycin resistance gene. (a) Cells expressing the chimeric type I erythropoietin (Epo) receptor fused to the bait at the carboxyl terminus (CR-bait) are generated by Flp recombinase-assisted integration followed by hygromycin selection. (b) Hygromycin-resistant cells are subsequently infected for 24-48 hours with a retrovirus encoding gp130 (part of the signaling cascade) fused to the prey protein (gp130-prey). (c) Cells are treated with erythropoietin for another 24-48 hours before puromycin selection. If the bait and prey proteins interact, binding of the ligand to the receptor can induce a signaling cascade through the Janus kinase (Jak), leading to the induction of the puromycin-resistance marker (Puro^R) and thus to growth of the cells in puromycin-containing selective medium. Prey proteins can be rapidly identified by direct amplification of their transcripts from lysed cell colonies by reverse-transcriptase PCR. Adapted with permission from Eyckerman S, Verhee A, der Heyden JV, Lemmens I, Ostade XV, Vandekerckhove J, Tavernier J: Nat Cell Biol 2001, 3:1114-1119.