A ZFN pair was designed to target exon 8 of pig GGTA1 gene. LDC were transfected with GGTA1 ZFN plasmids. SURVEYOR assay was used to evaluate the ZFN activity in LDC. GGTA1 gene knockout cells (GTKO) were obtained by counter-selection and used as nuclear donors for SCNT. The cloned fetuses and piglets were characterized by DNA sequencing. Expression of ¦Á-Gal epitope was further examined by flow cytometry and confocal microscopy.
SURVEYOR assay revealed 6.48 % ZFN activity in LDC. GTKO cells were obtained by counter-selection 10 d after ZFN transfection. A total of six fetuses and 13 piglets were produced by SCNT. All fetuses and piglets had biallelic mutations in the ZFN targeted region and were negative for ¦Á-Gal epitope.
Biallelic GGTA1 gene disruption in LDC was generated efficiently by ZFN. GTKO fetuses were produced from ZFN-treated LDC by SCNT. GTKO piglets were obtained by SCNT of ZFN-treated LDC or recloning of fetal fibroblasts from GTKO fetuses. With longer lifespan and robust growth rate, LDC has the potential to endure multiple genetic modifications in?vitro without going to SCNT, which could accelerate the production of genetically modified pig organs for xenotransplantation.