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

Authors:
Koo OJ, Park SJ, Lee C, Kang JT, Kim S, Moon JH, Choi JY, Kim H, Jang G, Kim JS, Kim S, Lee BC.
In:
Source: Asian J Androl
Publication Date: (2014)
Issue: 27(3): 324-9
Research Area:
Stem Cells
Gene Expression
Basic Research
Cells used in publication:
Fibroblast, pig
Species: porcine
Tissue Origin: dermal
Platform:
4D-Nucleofector® X-Unit
Experiment
fetal pig fibroblasts (2x106 cells) were nucleofected using a 100 µL Nucleocuvette, CA137 program code, in an Amaxa 4D-Nucleofector (Lonza, P3 Primary Cell 4D-Nucleofector X Kit L) with a total of 45 µg plasmid DNA at a 2:2:1 weight ratio (plasmid encoding a left ZFN: plasmid encoding a right ZFN: eGFP reporter or H-2Kk reporter).
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.