Figure 1: Strategy for converting hSIRPA-BAC DNA into a piggyBac transposon.

(A) Diagram illustrating the strategy used for retrofitting hSIRPA-BAC DNA (RP11-887J4) with piggyBac TIR elements. 5′ TIR (green) and 3′ TIR (orange) elements were sub-cloned into pUC19 vector backbone with spectinomycin resistance gene (purple), and 50 bp homology arm sequences (red) used for replacing the chloramphenicol resistance gene in the BAC vector backbone via recombineering technology. The diagram also indicates that the genomic DNA insert in the RP11-887J4 BAC is 176,233 bps, covering the SIRPA genic region, on chromosome 20 between 1,842,086-2,018,318. (B) The green arrows indicate the primer pairs used to verify hSIRPA-BAC retrofitting after the recombineering process. (C) A schematic diagram describing the transpositioning strategy of hSIRPA-BAC retrofitted with TIR elements mediated by piggyBac transposase. Illustration (i) shows the retrofitted BAC DNA. Illustrations (ii) and (iii) show the process by which the piggyBac transposase proteins bind to the TIR sequences, initiating nicking of the DNA strands, allowing 3′ hydroxyl group at both ends of the transposon to hydrophilic attack the flanking TTAA sequence and freeing the BAC from the spectinomycin resistance gene by forming hairpin structure at the TIR ends. Once the BAC DNA is released from spectinomycin resistance gene, illustration (iv) shows repairing of the linearized BAC DNA by ligating into the complementary TTAA overhangs in the genomic DNA through the mediation of the piggyBac transposase proteins.