title: Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity.
abstract: OBJECTIVE
  Production of alpha-1,3-galactosyltransferase (αGT)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. HeterozygousαGT knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produceαGT-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts.
  
  
  METHODS
  Miniature pig fibroblasts were transfected withαGTgene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygousαGTgene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-α-1,3-galactose, an epitope produced byαGT. Using magnetic activated cell sorting, cells with monoallelic disruption ofαGT were removed. Remaining cells with LOH carrying biallelic disruption ofαGT were used for the second round NT to produce homozygousαGTgene-targeted piglets.
  
  
  RESULTS
  Monoallelic mutation ofαGTgene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygousαGTgene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygousαGT knockout piglets.
  
  
  CONCLUSIONS
  The present study demonstrates that the time required for the production ofαGT-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial length of time for breeding.
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea.|Institute of Green Bioscience and Technology, Seoul National University, Pyeongchang 25354, Korea.
authors.forename: Young June
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea.
authors.forename: Kwang Sung
authors.initials: KS
authors.lastname: Ahn
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea.
authors.forename: Minjeong
authors.initials: M
authors.lastname: Kim
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea.
authors.forename: Min Ju
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea.
authors.forename: Jin Seop
authors.initials: JS
authors.lastname: Ahn
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea.
authors.forename: Junghyun
authors.initials: J
authors.lastname: Ryu
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea.
authors.forename: Soon Young
authors.initials: SY
authors.lastname: Heo
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea.
authors.forename: Sang-Min
authors.initials: SM
authors.lastname: Park
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea.
authors.forename: Jee Hyun
authors.initials: JH
authors.lastname: Kang
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea.
authors.forename: You Jung
authors.initials: YJ
authors.lastname: Choi
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea.|Institute of Tissue Regeneration Engineering, Dankook University, Cheonan 31116, Korea.|Department of Physiology, Dankook University School of Medicine, Cheonan 31116, Korea.
authors.forename: Hosup
authors.initials: H
authors.lastname: Shim
keywords: 3-galactosyltransferase; Alpha-1; Gene Targeting; Loss of heterozygosity; Nuclear Transfer; Pig