Monitoring bovine fetal fibroblast reprogramming utilizing a bovineNANOGpromoter-driven EGFP reporter system

Abstract

NANOG is an essential transcription factor involved in the proliferation and maintenance of embryonic stem cells (ESC) and reprogramming of somatic cells to a pluripotent state. Oct4 and Nanog promoter-driven enhanced green fluorescent protein (EGFP) reporters have been employed for establishing lines of induced pluripotent stem cells (iPSC) from mouse, human, and pig. In ruminants, including cattle, in which no fully validated ESC lines have been established, iPSC generated by reprogramming somatic cells to an ESC-like state may prove useful in the production of genetically modified livestock. In this study, utility of the bovine NANOG reporter was tested for use with cattle. Seven proximal bovine NANOG promoter fragments of different size were fused to the LUC gene, and were tested in mouse ESC lines using a dual-luciferase assay. Three of the bovine NANOG promoters, 315 bp (-134/+181), 446 bp (-265/+181), and 1,100 bp (-919/+181), were fused to a nuclear localized signal EGFP reporter gene. The fidelity of these constructs was analyzed by transfection into mouse ESC and bovine fetal fibroblasts (bFFs), and subsequent reprogramming of the bFF. Fusion of the transgenic bFF with human teratocarcinoma (NTERA2) cells induced nuclear expression of the EGFP reporter. Similarly, bFF-derived somatic cell nuclear transfer (SCNT) embryos expressed EGFP in a stage- and location-appropriate manner. Following reprogramming of transgenic bFFs for 10 days with an Oct4-Sox2-Klf4-cMyc vector, iPSC expressed EGFP and alkaline phosphatase. These results indicate that NANOG reporters can be used to monitor nuclear reprogramming of bFFs and to distinguish cell allocation in SCNT-derived embryos.