general characteristics of a primed pluripotent state in terms of marker gene expression, ability to differentiate in vitro, and normal karyotype. The lentiviral construct used in this 2199952 study is pLL3.7 consisted of EGFP as a marker gene with the CMV promoter and cis-acting elements including FLAP, WRE, 39SIN-LTR and multiple cloning site for shRNA. The reason this construct was chosen is as follows: 1) This vector has been proven to work efficiently in mouse embryos, primary cells and embryonic stem cell lines, and transgenic mice have been generated using mouse ESCs and embryos transfected with this vector. 2) In previous transfection studies involving porcine pluripotent cells, the CMV promoter was used for the establishment of transgene-expressed cell lines, these resulting transduced cell lines stably expressed the transgene for more than 90 months. When EpiSC-like pESCs were transfected with this vector under various MOIs, cytotoxicity was not detected up to a MOI of 75. At a MOI of 100, however, cytotoxicity occurred and reduced the number of EGFP-expressing cells. Moreover, EGFP expression was stronger at the edge of the colonies, likely because of metabolic upregulation and high cell density due to proliferation of dividing cells at the edge of the colonies. To examine the decline in EGFP expression during extended 26507655 culture, we assessed whether transgene expression was affected by DNA methylation or histone acetylation, which are known to epigenetically regulate gene expression. Methylated cytosine in DNA represses gene expression via recruitment of proteins associated with heterochromatin such as MeCP2. Acetylation on histone tails activates gene expression by increasing the negative charge of histones. Bisulfite sequencing data indicated that the DNA methylation level of the promoter region and expression of the transgene were negatively correlated because DNA methylation in the CMV promoter region increased with a concomitant decrease in EGFP expression. However, methylation levels of the EGFP region were not related to transgene expression, in contrast to a previous report involving transgenic animals. The EGFP region was hypomethylated regardless of EGFP expression. The expression level of transgenes is dependent upon the vector construct, transfection methods used and cell types. It is therefore important that the characterization of vector activities in various cell types is given attention. In the case of the CMV promoters used in this study, differences in expression patterns have been Triptolide web reported in several papers via the characterization of the transcriptional activities in ESCs. Some studies showed that the CMV promoter is active in human or mouse ESCs during long term culture. Conversely, other studies have reported that CMV-driven transgene expressions are rapidly downregulated or inactive in human or mouse ESCs. Silenced transgenes were not reactivated during differentiation. In porcine studies, CMV-driven 
expression of GFP was stably expressed during the propagation of pESCs , and transgenic porcine embryonic germ cell lines were successfully established using a CMV-EGFP construct. Our data has shown that the CMV-driven expression of GFP was progressively downregulated by DNA methylations and reactivated during differentiation by transacting factors without changes of DNA methylation levels. This result is consistent with previous findings of the latter cases in humans and mice with the exception of the upregulation o
