Cloning and Characterization of 3.1kb Promoter Region of the Oct4 Gene from the Fischer 344 Rat

Abstract

Here, the role of methylation in regulation of rat Oct4 gene was evaluated during embryonic development, in adult tissues and in embryo-derived cells. First, the region 3.1 kb upstream to the rat Oct4 ATG site was cloned and sequenced. The rat Oct4 upstream sequence was similar to that in bovine, mouse and human with two upstream elements: proximal (PE) and distal enhancers (DE) and four homology conserved regions (CR1-4). The conserved regions in the rat have 69% - 96% homology with bovine, human, mouse sequences. Next, the methylation pattern in the promoter was determined during embryonic development, in adult tissues, in rat embryonic stem cell (ESC)-like cells and umbilical cord-derived cells (the feeder for ESC-like cells) using the bisulfite method and DNA sequencing. The promoter was methylated in adult and fetal tissues, and in days post coitus (DPC) 10.5 and 12.5 embryos and hypomethylated in DPC4.5 embryos and in rat ESC-like cells. The expression of Oct4 was evaluated by qRT-PCR. DPC4.5 embryos and rat ESC-like cells had higher expression of the Oct4 gene compared to DPC10.5 and 12.5 embryos, adult tissues and embryoid bodies derived from rat ESC-like cells. Thus, the methylation status correlated with the qRT-PCR results. These results indicate that the rat Oct4 3.1kb promoter region is organized and contains transcription binding and regulatory sites similar to those described for bovine, mouse and human. The rat Oct4 promoter is methylated during embryonic development after 4.5 DPC and during differentiation of rat ESC-like cells to embryoid bodies.

Embryonic Stem Cells Induce Pluripotency in Somatic Cell Fusion through Biphasic Reprogramming

“…The genomic regions of Oct4 targeted for bisulfite sequencing, CR1 and CR4, were chosen based on cross-species conservation and correlation between methylation and expression as previously defined (He et al, 2009; Imamura et al, 2006). …”

Inhibition of Protein Kinase C Signaling Maintains Rat Embryonic Stem Cell Pluripotency*

“…Potential-To determine whether PKCi could maintain previously established rESC lines in an undifferentiated state, we cultured an F344 rESCs line that expresses EGFP under the control of a 3.1-kb portion of the rat Oct4 promoter/enhancer region (4,17). We found that PKCi culture efficiently maintains undifferentiated colony morphology and OCT4-EGFP reporter expression of established rESC lines (Fig.…”

Derivation and Characterization of Embryonic Stem Cells Lines Derived from Transgenic Fischer 344 and Dark Agouti Rats

“…Pbgd was used for quantitative (q)RT-PCR normalization via the DC t method [22]. Reactions for qRT-PCR were run in duplicates and averaged and were also run using 2 or 20 ng per tube cDNA loading concentrations.…”

“…Plasmid N1-Oct4-GFP contains a 3.1 kb portion of the rat Oct4 promoter including both the proximal enhancer and a portion of the distal enhancer. The Oct4 promoter and the rat Oct4 ATG site was inserted 5' to GFP, polyA sequence [22]. pN1-Oct4-GFP also contains a neomycin/kanamycin selection cassette under a separate SV40 promoter.…”

“…Here, we transfected rat ESCs with a plasmid made by fusing a 3.1 kb fragment of the rat Oct4 promoter that 1584 HONG, HE, AND WEISS contains the proximal enhancer and some of the distal enhancer [22] immediately upstream of a eGFP and poly A and neomycin resistance cassette (Fig. 5A).…”