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.

Effects of proteinate complex zinc on growth performance, hepatic and splenic trace elements concentrations, antioxidative function and immune functions in weaned piglets

“…Zinc (Zn) is an important trace element involved in forming more than 300 kinds of metalloenzymes that affect biochemical processes of the whole body. Known functions of Zn include: regulating immune response, improving gastrointestinal digestion and interacting with the nervous system [ 1 ]. Many studies demonstrated Zn deficiency is common in several psychiatric disorders including induction of Alzheimer’s disease [ 2 ].…”

Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

“…As the “editing windows” of the Target-AID and BE systems are different, these two base-editing systems may be used complementarily for complex porcine genome engineering. Furthermore, in combination with zygote electroporation [ 31 ] or somatic cell nuclear transfer [ 32 ], it is expected that base-edited pigs can be generated using the Target-AID system. Particularly, for generating PERV inactivated pigs, ribonucleoprotein (RNP) based Target-AID is recommended to avoid safety issue in clinical xenotransplantation.…”

Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

“…As the "editing windows" of the Target-AID and BE systems are different, these two base-editing systems may be used complementarily for complex porcine genome engineering. Furthermore, in combination with zygote electroporation [31] or somatic cell nuclear transfer [32], it is expected that base-edited pigs can be generated using the Target-AID system.…”