Related Experiment Video
Updated: Sep 15, 2025

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Published on: May 30, 2012
Regulation of PKCi-mediated pluripotency and gene expression by polycomb complex 1 in mouse embryonic stem cells
Fangfang Wu1, Zhihui Liu1, Xiaohu Wu1
1Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University Nanjing 210046, Jiangsu, China.
Objectives:
Epigenetic modification of Polycomb repressive complex 1 (PRC1) plays an important role in regulating the pluripotency and self-renewal of embryonic stem cells (ES). Inhibition of protein kinase C (PKCi) can capture and maintain the pluripotency of mouse ES (mES). Here, we characterized the dynamic expression pattern of PRC1 and its mechanism of action in PKCi-derived mES (PKCi-mES).
Methods:
The expression of PRC1 components in PKCi-mES, 2iL-derived mES (2iL-mES), and mouse embryonic fibroblasts (MEF) was comparatively analyzed using Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot to identify differentially expressed subunits. Subsequent knockdown of these differential components was performed to examine their effects on cellular differentiation status, along with the expression of pluripotency genes and differentiation genes.
Results:
We found that PKCi-mES showed high expression of the PRC1 functional catalytic subunit RING1B, canonical PRC1-specific component CBX7, and non-canonical PRC1 specific component RYBP at both gene and protein levels in comparison with control. Knocking down Ring1b and Cbx7 accelerated the differentiation of PKCi-mES and reduced the expression of core pluripotency genes and naïve marker genes. Genes associated with mesoderm, ectoderm, and endoderm differentiation were broadly upregulated after Ring1b knockdown, while Cbx7 knockdown upregulated the expression of Cbx8 and some ectoderm genes but downregulated the expression of some endoderm and mesoderm genes.
Conclusions:
These results indicate that PKCi activates both the canonical and non-canonical PRC1 pathways, finely regulates the expression of mES pluripotent and developmental genes, and helps maintain mES in a poised state between self-renewal and lineage commitment.
More Related Videos
07:18A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening
Published on: May 12, 2017
11:53An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C
Published on: June 1, 2018
Related Concept Videos
Combinatorial Gene Control
The expression of more than 30,000 genes is controlled by approximately 2000-3000 transcription factors. This is possible because a single transcription factor can recognize more than one regulatory sequence. The specificity in gene...
Somatic to iPS Cell Reprogramming
Maintenance of the ES Cell State
Chromatin Modification in iPS Cells
Compact chromatin makes reprogramming difficult. Enzymes, such as histone demethylases and acetyltransferases, are often added during reprogramming to loosen the chromatin, making the DNA more accessible to transcription factors. Molecules that inhibit histone...
Induced Pluripotent Stem Cells
Somatic...
Methods of Nuclear Reprogramming