Figure 2.
Summary of the somatic cell nuclear transfer (SCNT) approach used to produce CFTR gene-targeted ferrets. Primary fetal ferret fibroblasts are infected with rAAV-targeting virus and subsequently cloned by limiting dilution under G418 selection. Following replica plating and PCR screening in 96-well plates, cells are minimally expanded and then used for SCNT. Embryo fibroblasts (21 days) are then derived under G418 selection and screened by Southern blotting for the CFTR gene-targeting event. Rejuvenated fibroblasts containing the CFTR-targeted allele are then used for a second round of SCNT to clone live ferrets.