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Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System

  • Koo, Ok Jae (Laboratory Animal Research Center, Samsung Biomedical Research Institute) ;
  • Park, Sol Ji (Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University) ;
  • Lee, Choongil (National Creative Research Initiatives Center for Genome Engineering, Department of Chemistry, Seoul National University) ;
  • Kang, Jung Taek (Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University) ;
  • Kim, Sujin (Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University) ;
  • Moon, Joon Ho (Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University) ;
  • Choi, Ji Yei (Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University) ;
  • Kim, Hyojin (National Creative Research Initiatives Center for Genome Engineering, Department of Chemistry, Seoul National University) ;
  • Jang, Goo (Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University) ;
  • Kim, Jin-Soo (National Creative Research Initiatives Center for Genome Engineering, Department of Chemistry, Seoul National University) ;
  • Kim, Seokjoong (ToolGen Inc.) ;
  • Lee, Byeong-Chun (Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University)
  • Received : 2013.08.07
  • Accepted : 2013.09.25
  • Published : 2014.03.01

Abstract

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells ($RFP^+/eGFP^+$) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

Keywords

Knockout Pig;Reporter Based Enrichment;Somatic Cell Nuclear Transfer;Zinc Finger Nuclease

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