The Oct4 promoter-EGFP transgenic rabbit: a new model for monitoring the pluripotency of rabbit stem cells

Abstract

The rabbit has long been used as a laboratory animal model for developing reproductive and stem cell-related technologies, as well as for studying human disease. The Oct4 transcription factor plays a crucial role in the maintenance and regulation of pluripotency in embryos and stem cells. We constructed a reporter plasmid containing the gene encoding the enhanced green fluorescent protein (EGFP) under the control of the rabbit Oct4 promoter (prOG) and transfected it into E14 mouse stem cells and rabbit ESCs. In addition, prOG transgenic fibroblasts were derived and prOG transgenic rabbits were produced by somatic cell nuclear transfer (SCNT). The pattern of expression of ectopic EGFP was similar in E14 mouse stem cells whether under the control of the rabbit (prOG) or mouse Oct4 promoter (pmOG). EGFP expression was observed in rabbit ESCs following prOG transfection. Both prOG transgenic SCNT embryos and F1 prOG transgenic embryos derived from adult transgenic rabbits expressed green fluorescence at the morula and blastocyst stages. EGFP was clearly detected in gonads isolated from fetuses at 27 dpc. The prOG transgenic rabbit represents a new model for studying the derivation and maintenance of rabbit pluripotent cells, and for investigating rabbit embryo development.

Establishment of a Rabbit Oct4 Promoter-Based EGFP Reporter System

“…We found that 44.4% of the reconstructed blastocysts expressed EGFP which is consistent with other reports on rabbits and pigs using Oct4-EGFP reporter systems [25], [26]. The rate of embryos expressing EGFP, instead of blastocyst rate, may represent the actual reprogramming efficiency of SCNT.…”

“…Yin et al [25] have recently conducted a similar study. The following are the key differences between our work and that of Yin et al Firstly, the sequence of the Oct4 promoter in our work included not only the DE, the PE and the basic promoter region but also the whole region of the first exon to simulate endogenous Oct4 expression for the regulator may also in the exons [29].…”

Beyond the mouse: non-rodent animal models for study of early mammalian development and biomedical research

“…In mice, genetic modifications can be effectively introduced by homologous recombination into ESCs (Bronson and Smithies, 1994), however, germline competent rabbit ESC lines are not yet available. Previously, reporter lines such as Oct4-EGFP have been created by transfection of somatic cells and subsequent somatic cell nuclear transfer (Quan et al, 2014, Yin et al, 2013. Although effective, this method is very time-and labour-consuming.…”

Cloning and characterization of rabbit POU5F1, SOX2, KLF4, C-MYC and NANOG pluripotency-associated genes

“…Particularly, the expression of POU5F1 could be followed in developing embryos of POU5F1 promoter-EGFP transgenic rabbits (Yin et al, 2013). In another study, the expression level of POU5F1, SOX2, NANOG and KLF4 was measured by quantitative PCR during rabbit preimplantation development (Henderson et al, 2014).…”