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

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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
JournalJournal of Surgical Research
Volume181
Issue number1
DOIs
StatePublished - May 1 2013

Fingerprint

Galactosyltransferases
Zinc Fingers
Swine
Liver
Genes
Gene Knockout Techniques
Fetus
Epitopes

Keywords

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

ASJC Scopus subject areas

  • Surgery

Cite this

Biallelic knockout of the α-1,3 galactosyltransferase gene in porcine liver-derived cells using zinc finger nucleases. / Li, Ping; Estrada, Jose L.; Burlak, Christopher; Tector, A. Joseph.

In: Journal of Surgical Research, Vol. 181, No. 1, 01.05.2013.

Research output: Contribution to journalArticle

Li, Ping ; Estrada, Jose L. ; Burlak, Christopher ; Tector, A. Joseph. / Biallelic knockout of the α-1,3 galactosyltransferase gene in porcine liver-derived cells using zinc finger nucleases. In: Journal of Surgical Research. 2013 ; Vol. 181, No. 1.
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title = "Biallelic knockout of the α-1,3 galactosyltransferase gene in porcine liver-derived cells using zinc finger nucleases",
abstract = "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.",
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AU - Li, Ping

AU - Estrada, Jose L.

AU - Burlak, Christopher

AU - Tector, A. Joseph

PY - 2013/5/1

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N2 - 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.

AB - 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.

KW - Fetal liver-derived cells

KW - Genetic modification

KW - Somatic cell nuclear transfer

KW - Xenotransplantation

KW - Zinc finger nucleation

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