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

• Published in: Asian-australasian journal of animal sciences Vol. 27; no. 3; pp. 324 - 329
• Main Authors: Koo, Ok Jae, Park, Sol Ji, Lee, Choongil, Kang, Jung Taek, Kim, Sujin, Moon, Joon Ho, Choi, Ji Yei, Kim, Hyojin, Jang, Goo, Kim, Jin-Soo, Kim, Seokjoong, Lee, Byeong-Chun
• Format: Journal Article
• Language: Korean
• Published: 2014
• Subjects: Knockout Pig; Somatic Cell Nuclear Transfer; Zinc Finger Nuclease; Reporter Based Enrichment
• ISSN: 1011-2367; 1976-5517

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.