Biallelic knockout of the α-1,3 galactosyltransferase gene in porcine liver-derived cells using zinc finger nucleases

Ping Li, Jose L. Estrada, Christopher Burlak, A. Joseph Tector

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32 Scopus citations


Background: Genetic modification of the pig has been hampered by inefficiency of homologous recombination and unavailability of pig embryonic stem cells. Engineered zinc finger nuclease (ZFN)-mediated genetic modification in somatic cells combined with somatic cell nuclear transfer (SCNT) technology provides a new approach for targeted modification in pig genome. In this study, we used a ZFN pair to disrupt porcine α-1,3, galactosyltransferase (GGTA1) gene in liver-derived cells (LDC). ZFN-treated LDC were used as nuclear donors to produce fetuses and piglets via SCNT. All cloned fetuses and piglets showed biallelic knockout of GGTA1 gene. Materials and methods: 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 α-Gal epitope was further examined by flow cytometry and confocal microscopy. Results: 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 α-Gal epitope. Conclusions: 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.

Original languageEnglish (US)
Pages (from-to)E39-E45
JournalJournal of Surgical Research
Issue number1
StatePublished - May 1 2013

Bibliographical note

Funding Information:
This study was supported by IU Health Transplant Institute . The authors thank Amanda G. Clark, Fuqin Yin, and Bess Ivary for technical assistance, and MRI (Methodist Research Institute) and LARC (Laboratory Animal Research Center) staff for taking care of the animals. This investigation utilized a facility constructed with support from Research Facilities Improvement Program grant number C06RR10601-01 from the National Center for Research Resources, National Institutes of Health .


  • Fetal liver-derived cells
  • Genetic modification
  • Somatic cell nuclear transfer
  • Xenotransplantation
  • Zinc finger nucleation


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