Figure 7.

Schematic of VHL gene reconstitution by serial integration into the IIS-alphoid^tetO-HAC. The VHL gene is located on chromosome 3 (positions 10137959–10154492; GHCH38/hg38). The gene contains three exons. Mutations in the gene are associated with the Von Hippel-Lindau (VHL) syndrome, a dominantly inherited hereditary cancer syndrome predisposing to a variety of malignant and benign tumors of the eye, brain, spinal cord, kidney, pancreas, and adrenal glands. (a) Construction of Type I vectors carrying genomic fragments DNA1 and DNA3,encompassing exon 3 and exon 1, respectively, and Type II vector carrying a genomic DNA2 fragment encompassing exon 2 of the VHL gene. DNA1 (exon 3) and DNA3 (exon 1) were inserted into the AscI/NotI sites of the Type I carrier vector A167. DNA2 (exon 2) was inserted into the AscI/NotI sites of the Type II carrier vector A169. (b) Three rounds of insertion of the VHL fragments into the IIS-alphoid^tetO-HAC carrying the integration platform cassette are shown. Round 1: A diagnostic PCR for DNA1 insertion was performed with primers R3/B678. Round 2: A diagnostic PCR for DNA2 insertion was performed with primers F23/R23. Round 3: A diagnostic PCR for DNA3 insertion was performed with diagnostic primers F12/R12. The position of the primer pairs are shown. (c) Representative images of how cell fluorescence changes after each round of DNA insertion are shown.