Generation and characterization of a novel knockin minipig model of Hutchinson-Gilford progeria syndrome

a Genetic engineering procedure for generating Hutchinson-Gilford progeria syndrome (HGPS) knockin Yucatan minipig fibroblasts by CRISPR/Cas9-mediated homologous recombination. The area of hybridization for sgRNA1 in genomic DNA is indicated by a yellow arrowhead, the area of recombination between genomic DNA and donor DNA template is indicated by green lines, and the single C？>？T base mutation in the donor DNA template is indicated by a red T in exon 11. LHA and RHA respectively indicate the left and right homology arms surrounding the C？>？T mutation (not drawn to scale). For an overview of the whole method to generate HGPS minipigs and its efficiency see Supplementary Fig. S2. For detailed information of molecular tools see Supplementary Table S1 and 2. b Genomic LMNA exon 11 sequence of wild-type (WT) minipig fibroblasts and the generated heterozygous HGPS knockin fibroblast clone. Note that WT cells are homozygous for the C nucleotide in position c.1824 (blue arrowhead), and HGPS cells harbor both the WT C allele and the mutant T allele (red arrowhead). c Representative photographs showing the normal appearance of HGPS minipigs at birth and the severe premature-aging external phenotype at 5.5–6 months of age. Note that pictures were not taken at the same distance. See also Supplementary Fig. S3 and Movies S1-5. d HGPS Yucatan minipigs experience difficulty to thrive. The graph shows body weight/age curves (n？=？6 WT; n？=？10 HGPS). e HGPS minipigs die prematurely. Green dots in the Kaplan-Meier survival curve are censored data (1 HGPS minipig died during anesthesia induction in preparation for imaging tests and another was euthanized due to humane end-point criteria