Skip to main content
Journal cover image

Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types.

Publication ,  Journal Article
Rodriguez, RT; Velkey, JM; Lutzko, C; Seerke, R; Kohn, DB; O'Shea, KS; Firpo, MT
Published in: Exp Biol Med (Maywood)
November 2007

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.

Duke Scholars

Altmetric Attention Stats
Dimensions Citation Stats

Published In

Exp Biol Med (Maywood)

DOI

ISSN

1535-3702

Publication Date

November 2007

Volume

232

Issue

10

Start / End Page

1368 / 1380

Location

Switzerland

Related Subject Headings

  • Transfection
  • Reverse Transcriptase Polymerase Chain Reaction
  • RNA Interference
  • Octamer Transcription Factor-3
  • Molecular Sequence Data
  • Immunohistochemistry
  • Humans
  • Genes, Reporter
  • Embryonic Stem Cells
  • Electroporation
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Rodriguez, R. T., Velkey, J. M., Lutzko, C., Seerke, R., Kohn, D. B., O’Shea, K. S., & Firpo, M. T. (2007). Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types. Exp Biol Med (Maywood), 232(10), 1368–1380. https://doi.org/10.3181/0703-RM-63
Rodriguez, Ryan T., J Matthew Velkey, Carolyn Lutzko, Rina Seerke, Donald B. Kohn, K Sue O’Shea, and Meri T. Firpo. “Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types.Exp Biol Med (Maywood) 232, no. 10 (November 2007): 1368–80. https://doi.org/10.3181/0703-RM-63.
Rodriguez RT, Velkey JM, Lutzko C, Seerke R, Kohn DB, O’Shea KS, et al. Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types. Exp Biol Med (Maywood). 2007 Nov;232(10):1368–80.
Rodriguez, Ryan T., et al. “Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types.Exp Biol Med (Maywood), vol. 232, no. 10, Nov. 2007, pp. 1368–80. Pubmed, doi:10.3181/0703-RM-63.
Rodriguez RT, Velkey JM, Lutzko C, Seerke R, Kohn DB, O’Shea KS, Firpo MT. Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types. Exp Biol Med (Maywood). 2007 Nov;232(10):1368–1380.
Journal cover image

Published In

Exp Biol Med (Maywood)

DOI

ISSN

1535-3702

Publication Date

November 2007

Volume

232

Issue

10

Start / End Page

1368 / 1380

Location

Switzerland

Related Subject Headings

  • Transfection
  • Reverse Transcriptase Polymerase Chain Reaction
  • RNA Interference
  • Octamer Transcription Factor-3
  • Molecular Sequence Data
  • Immunohistochemistry
  • Humans
  • Genes, Reporter
  • Embryonic Stem Cells
  • Electroporation