Fig. 1

Development of NF1 minipigs. a Human exon 39 and swine exon 41 of the NF1 gene show 100% amino acid homology. A pair of TALENs was designed to bind swine NF1 exon 41 in the region of R1947. The entire exon is not shown; gray letters, differences in nucleotide sequences; bold letters, TALEN-binding sites; blue letters, amino acid sequence. b The NF1^R1947* allele was engineered into the swine genome using homologous recombination (HR) of a 90mer HDR oligonucleotide containing a HindIII RFLP site to allow for facile analysis of HR-positive cells. Bold letters, TALEN-binding sites; red italicized nucleotides, novel stop codon (R1947*); black italicized nucleotides, added nucleotides; blue letters, amino acids; red letters, amino acid code resulting from frameshift. c Minipig embryonic fibroblasts were transfected with TALENs and HDR oligonucleotides and individual cells were isolated and grown as single cell-derived colonies. These colonies were assayed for incorporation of the RFLP site by HindIII restriction enzyme digest followed by gel electrophoresis. The closed arrowhead denotes the wild-type allele and the open arrowhead denotes the RFLP allele. Several clones were TOPO cloned and sequenced to confirm the presence of both the wild-type allele and the NF1^R1947* allele (clone 89 is shown as an example). Three to five sequence-confirmed NF1^R1947*/+ clones were pooled and underwent chromatin transfer to produce F0 NF1 male minipigs that were subsequently bred to wild-type females to produce F1 minipigs. d A representative example of a CALM (white arrow) seen in an NF1 minipig at 5 months of age. e An example of multiple CALMs seen in an NF1 minipig at 16 months of age. Scale bar, 2 cm. f H&E staining of adjacent normal NF1 minipig skin and wild-type minipig skin (shown here) shows vascular beds (squares) and melanin but no melanin deposits. Scale bar, 200 µM. g H&E staining of CALMs shows melanin deposits in the basal layer of the epidermis (black arrowheads). Scale bar, 200 µM