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Manipulation of OCT4 Levels in Human Embryonic Stem Cells Results in Induction of Differential Cell Types
- Source :
- Experimental Biology and Medicine. 232:1368-1380
- Publication Year :
- 2007
- Publisher :
- SAGE Publications, 2007.
-
Abstract
- To fully understand self-renewal and pluripotency and their regulation in human embryonic stem cells (hESCs), it is necessary to generate genetically modified cells and analyze the consequences of elevated and reduced expression of genes. Genes expressed in hESCs using plasmid vectors, however, are subject to silencing. Moreover, hESCs have a low plating efficiency when dissociated to single cells, making creation of subcloned lines inefficient. In addition to overexpression experiments, it is important to perform loss-of-function studies, which can be achieved rapidly using RNA interference (RNAi). We report stable long-term expression of enhanced green fluorescent protein (eGFP) in hESCs using a lentiviral vector, and establishment of an eGFP-expressing subline (RG6) using manual dissection. To demonstrate the efficacy of RNAi in hESCs, an RNAi expression vector was used to achieve reduced expression of eGFP in hESCs. To evaluate the role of OCT4 in the regulation of hESC self-renewal and differentiation, a vector expressing a hairpin RNA targeting endogenous expression of OCT4 was constructed. In a novel experiment in hESCs, the OCT4 cDNA sequence was cloned into an expression vector to allow for the transient upregulation of OCT4 in hESCs. The ability to manipulate levels of OCT4 above and below enodogenous levels allows the determination of OCT4 function in hESCs. Specifically, reduced expression of OCT4 in hESCs promoted upregulation of markers indicative of mesoderm and endoderm differentiation, and elevated levels of OCT4 in hESCs promoted upregulation of markers indicative of endoderm derivatives. Thus, both upregulation and downregulation of Oct4 in hESCs results in differentiation, but with patterns distinct from parallel experiments in mice.
- Subjects :
- 0301 basic medicine
Molecular Sequence Data
Biology
Transfection
General Biochemistry, Genetics and Molecular Biology
Cell Line
Small hairpin RNA
03 medical and health sciences
0302 clinical medicine
Downregulation and upregulation
Genes, Reporter
RNA interference
medicine
Humans
Embryonic Stem Cells
reproductive and urinary physiology
Gene knockdown
Expression vector
Base Sequence
Reverse Transcriptase Polymerase Chain Reaction
fungi
Cell Differentiation
equipment and supplies
Immunohistochemistry
Molecular biology
Embryonic stem cell
Electroporation
030104 developmental biology
medicine.anatomical_structure
030220 oncology & carcinogenesis
embryonic structures
RNA Interference
biological phenomena, cell phenomena, and immunity
Stem cell
Endoderm
Octamer Transcription Factor-3
Subjects
Details
- ISSN :
- 15353699 and 15353702
- Volume :
- 232
- Database :
- OpenAIRE
- Journal :
- Experimental Biology and Medicine
- Accession number :
- edsair.doi.dedup.....e230cebdc9013e95ef1370afedcf6c15