Figure 2.
FLPe-Mediated RMCE Allows Generation of Fully Recombined Lines, Free of Random Integration, with 100% Efficiency
(A) The RMCE donor vector pZ:F3-P CAGGS tdTPH-F (top) flanked by heterotypic FRT sequences (additional details in the Supplemental Information), original master cell line (MCL) and resulting RMCE line (RMCEL) are depicted. Red, blue, and green lines represent the 5′ internal, puromycin, and 3′ external Southern blot probes, respectively. Fragment sizes of DNA digested with NcoI (N) are indicated.
(B) Timeline of RMCEL generation (all PuroR/FIAUR cells) and selection program with FACS histograms representing the cassette exchange.
(C) PCR characterization of independent RMCELs using primer sets depicted in (A) for 5′/3′ JA of the MCL or RMCEL, 5′/3′ RI of the donor or FLPe-expressing vector. Wild-type (WT), MCL#13, and controls for random and FLPe integration (+RI and +FLPe) and no template control (NTC) samples were included.
(D) Southern blotting of hESC wild-type (WT) cells, MCLs and RMCELs.
(E) Teratoma formation assay of two RMCE lines.
Scale bars, 50 μm (black) and 100 μm (white).