Fig. 4
From: Genetically engineered minipigs model the major clinical features of human neurofibromatosis type 1
A subset of CALM-derived melanocytes and neurofibroma-derived Schwann cells show loss of heterozygosity. a The chromatogram trace peak area of T nucleotides at the level of the R1947* point mutation (CGA (R)→TGA (*)) relative to total trace peak area is displayed as the percentage of T nucleotides. Samples with >75% percent T nucleotides were considered to have undergone LOH. Samples with less than 75% T nucleotides were considered to have retained the wild-type allele. 4/8 melanocyte cultures derived from CALMs and 6/11 Schwann cell cultures from neurofibromas show LOH. In contrast, none of the six fibroblast cultures isolated from normal NF1 minipig skin (NF1+/− fibroblasts) show LOH. b Sanger sequencing shows various mechanisms of LOH. An example chromatograph of a wild-type allele and R1947* RFLP allele isolated by TOPO cloning and sequenced are shown at the top. Melanocytes isolated from a CALM from NF1 minipig 1359 show complete conversion to the RFLP allele as seen by PCR population allele sequencing. Melanocytes isolated from a CALM from NF1 minipig 1413 show LOH with incorporation of the NF1R1947* mutation, but not the HindIII RFLP site, as seen by TOPO cloning. c Five Schwann cell lines from NF1 minipig neurofibromas (tumors 1–5) with LOH show loss of neurofibromin protein expression. This western blot was cropped to improve the conciseness of the presentation. The full-length blot is presented in Supplementary Figure 7. This western blot is representative of three experiments. d Relative density quantification of neurofibromin protein expression to α-tubulin. Wild-type Schwann cells from wild-type minipig sciatic nerve, NF1+/− Schwann cells from NF1 minipig sciatic nerve, NF1−/− immortalized human NF1−/− Schwann cell line
