title: Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System.
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.
authors: Laboratory Animal Research Center, Samsung Biomedical Research Institute, Gyeonggi-do 440-746, Korea.
authors: Ok Jae
authors: OJ
authors: Koo
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea .
authors: Sol Ji
authors: SJ
authors: Park
authors: National Creative Research Initiatives Center for Genome Engineering, Department of Chemistry, Seoul National University, Seoul 151-742, Korea .
authors: Choongil
authors: C
authors: Lee
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea .
authors: Jung Taek
authors: JT
authors: Kang
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea .
authors: Sujin
authors: S
authors: Kim
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea .
authors: Joon Ho
authors: JH
authors: Moon
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea .
authors: Ji Yei
authors: JY
authors: Choi
authors: National Creative Research Initiatives Center for Genome Engineering, Department of Chemistry, Seoul National University, Seoul 151-742, Korea .
authors: Hyojin
authors: H
authors: Kim
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea . ; Emergence Center for Food-Medicine Personalized Therapy System, Advanced Institutes of Convergence Technology, Seoul National University, Gyeonggi-do 443-270, Korea .
authors: Goo
authors: G
authors: Jang
authors: National Creative Research Initiatives Center for Genome Engineering, Department of Chemistry, Seoul National University, Seoul 151-742, Korea .
authors: Jin-Soo
authors: JS
authors: Kim
authors: ToolGen Inc., Seoul 153-783, Korea .
authors: Seokjoong
authors: S
authors: Kim
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea .
authors: Byeong-Chun
authors: BC
authors: Lee
keywords: Knockout Pig; Reporter Based Enrichment; Somatic Cell Nuclear Transfer; Zinc Finger Nuclease