Figure 3: Integrity of transpositioned hSIRPA-BACs and copy number analysis.

(A) Graphic illustration showing the genomic DNA insert in RP11-887J4. The chromosomal locations of start and end points are indicated. Exon in the SIRPA gene are blocked in bold. Green arrows indicate the primer pairs (10) spanning along entire BAC at approximately 20 kb intervals, used to verify the presence of the full BAC in pups confirmed to have hSIRPA-BAC transpositioned. (B) PCR result showing that six pups (1.6, 5.4, 5.7, 8.1, 9.2 and 9.4) with transpositioned hSIRPA-BACs are confirmed to be positive for all ten primer pairs. Genomic DNAs from pups 2.3, 3.2 and 6.2 were used as negative controls (Fig. 2A). (C) PCR result two pups (4.2 and 5.5) that were found to be positive for the presence of the human SIRPA gene during the genotyping screening, but negative for piggyBac mediated transposition. The results show that pup 4.2 is positive only for primer pair 5, suggesting random insertion of a fragmented BAC DNA. Pup 5.5 is found to be positive for all ten primer pairs, suggesting random insertion of hSIRPA-BAC DNA. (D) Bar graph showing RT-qPCR result indicating the copy number of human SIRPA BAC in piggyBac transpositioned founders (1.6, 5.4, 5.7, 8.1, 9.2, 9.4), random insertion founder (5.5), two negative controls (#1, #2). RT-qPCR results were analyzed relative to the rat GAPDH, for which there are two copies in the genome.