title: Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals.
abstract: Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells.
authors.affiliation: Veterinary Reproduction and Obstetrics, Department of Large Animal Sciences, University of Copenhagen, DK1870 Frederiksberg C, Denmark. Electronic address: jbsecher@sund.ku.dk.
authors.forename: Jan O
authors.initials: JO
authors.lastname: Secher
authors.affiliation: Department of Animal Science, Aarhus University, DK8830 Tjele, Denmark.
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Institute for Farm Animal Genetics (FLI), Neustadt, Germany.
authors.forename: Stoyan
authors.initials: S
authors.lastname: Petkov
authors.affiliation: Department of Biomedicine, Aarhus University, DK8000 Aarhus C, Denmark.
authors.forename: Yonglun
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, DK1870 Frederiksberg C, Denmark.
authors.forename: Dong
authors.initials: D
authors.lastname: Li
authors.affiliation: Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, DK1870 Frederiksberg C, Denmark.
authors.forename: Vanessa J
authors.initials: VJ
authors.lastname: Hall
authors.affiliation: Veterinary Reproduction and Obstetrics, Department of Large Animal Sciences, University of Copenhagen, DK1870 Frederiksberg C, Denmark.
authors.forename: Mette
authors.initials: M
authors.lastname: Schmidt
authors.affiliation: Department of Animal Science, Aarhus University, DK8830 Tjele, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Callesen
authors.affiliation: Department of Clinical Medicine, Aarhus University, DK8200 Aarhus N, Denmark.
authors.forename: Jacob F
authors.initials: JF
authors.lastname: Bentzon
authors.affiliation: Department of Clinical Medicine, Aarhus University, DK8200 Aarhus N, Denmark.
authors.forename: Charlotte B
authors.initials: CB
authors.lastname: Sørensen
authors.affiliation: Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, DK1870 Frederiksberg C, Denmark.
authors.forename: Kristine K
authors.initials: KK
authors.lastname: Freude
authors.affiliation: Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, DK1870 Frederiksberg C, Denmark. Electronic address: poh@sund.ku.dk.
authors.forename: Poul
authors.initials: P
authors.lastname: Hyttel
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002460:Cell Line; D065150:Cellular Reprogramming; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary*; D004624:Embryo Transfer / Q000662:veterinary; D004622:Embryo, Mammalian / Q000502:physiology; D047108:Embryonic Development; D005260:Female; D005347:Fibroblasts / Q000502:physiology; D049452:Green Fluorescent Proteins; D008297:Male; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D039904:Pluripotent Stem Cells / Q000502:physiology*; D011247:Pregnancy; D013552:Swine / Q000235:genetics* / Q000502:physiology*
chemical_list: D049452:Green Fluorescent Proteins
keywords: Embryonic germ cell; Handmade cloning; Induced pluripotent stem cells; Pluripotency; Somatic cell nuclear transfer; Transgenic pigs