Transgene walking PCR assay. Starting from the NeoR gene, known to be present and expressed, 9 successive PCRs in sequence (W-01 to W-09) were designed to cover all transgene sequences to the LTR. PCR W-04 enabled detection of the spliced truncated HSV-tk gene. Different lengths enabled rapid determination of the 9 PCR products. In the case of negative PCRs (as illustrated for W-03 to W-04), secondary PCR products, obtained using transgene walking primers flanking the detected deletion area (del-F4 and del-R3 in this example), were cloned and sequenced.