Establishment of a Rabbit Oct4 Promoter-Based EGFP Reporter System

Abstract

Rabbits are commonly used as laboratory animal models to investigate human diseases and phylogenetic development. However, pluripotent stem cells that contribute to germline transmission have yet to be established in rabbits. The transcription factor Oct4, also known as Pou5f1, is considered essential for the maintenance of the pluripotency of stem cells. Hence, pluripotent cells can be identified by monitoring Oct4 expression using a well-established Oct4 promoter-based reporter system. This study developed a rabbit Oct4 promoter-based enhanced green fluorescent protein (EGFP) reporter system by transfecting pROP2-EGFP into rabbit fetal fibroblasts (RFFs). The transgenic RFFs were used as donor cells for somatic cell nuclear transfer (SCNT). The EGFP expression was detected in the blastocysts and genital ridges of SCNT fetuses. Fibroblasts and neural stem cells (NSCs) were derived from the SCNT fetuses. EGFP was also reactivated in blastocysts after the second SCNT, and induced pluripotent stem cells (iPSCs) were obtained after reprogramming using Yamanaka's factors. The results above indicated that a rabbit reporter system used to monitor the differentiating status of cells was successfully developed.

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

“…Moreover, the information provided here could be used instead of mouse or human sequences to construct vectors for more efficient reprogramming of rabbit somatic cells to pluripotency. In support of this, a POU5F1 promoter from rabbit was found to be more suitable than promoters from different species when a reporter system was constructed (Quan et al, 2014). Implementing the newly identified genes either as biomarkers or as reprogramming factors might eventually lead to the creation of stable rabbit pluripotent cell lines.…”

Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering

“…piPS-LCs were induced in accordance with a previously described protocol[ 16 ]. Briefly, human Oct4, Sox2, Klf4 and c-Myc were separately cloned into lentiviral vector FUGW (Addgene, 14883), and the constructs FUW-hOct4, FUW-hSox2, FUW-hKlf4 and FUW-hc-Myc were packaged into a virus in 293T cell lines by co-transfecting with auxiliary packaging vectors (psPAX2 and pMD2.G).…”

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

“…A number of transcription factors that have been associated with pluripotency in the mouse, human, and livestock embryos, are also expressed in the rabbit. OCT4 is expressed throughout the whole preimplantation period, from a zygote to an implanting blastocyst at 6 dpc, although the expression level decreases over time, as evidenced by mRNA expression analysis , Kobolak et al, 2009, Mamo et al, 2008, Táncos et al, 2015, immunofluorescent labelling (Canon et al, 2018, Yin et al, 2013, and expression of GFP reporters (Quan et al, 2014, Yin et al, 2013. Recent evidence has shown that the decrease might be related to progressive methylation of the 5' regulatory region of POU5F1 and its gradual repression in the rabbit TE (Canon et al, 2018).…”

“…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.…”