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Embryonic Stem Cells00:57

Embryonic Stem Cells

Embryonic stem (ES) cells were first discovered in mice in 1981 by Martin Evans. In 1998, James Thomson identified a method to isolate embryonic stem cells from humans. Human embryonic stem cells (hESCs) are obtained from 3-5 day old embryos that remain unused after an in vitro fertilization procedure.
ES cells are grown in a culture medium where they can divide indefinitely, creating ES cell lines. Under certain conditions, ES cells can differentiate, either spontaneously into a variety of...

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In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells
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Transient and stable transgene expression in human embryonic stem cells.

Chee-Gee Liew1, Jonathan S Draper, James Walsh

  • 1Centre for Stem Cell Biology, Department of Biomedical Science, Western Bank, University of Sheffield, Sheffield S10 2TN, United Kingdom.

Stem Cells (Dayton, Ohio)
|March 24, 2007
PubMed
Summary

Choosing the right promoter is critical for stable genetic manipulation of human embryonic stem cells. The CAG promoter with PyF101 enhancer offers superior long-term gene expression in hES cells compared to CMV, UbiC, and R26 promoters.

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Area of Science:

  • Stem Cell Biology
  • Molecular Biology
  • Genetic Engineering

Background:

  • Plasmid vectors are essential for genetic manipulation of human embryonic stem cells (hES).
  • Promoter selection significantly impacts transfection efficiency and transgene expression stability in hES cells.
  • Previous studies show variable transfection outcomes, necessitating a systematic promoter comparison.

Purpose of the Study:

  • To compare the efficacy of four different promoters for transient and stable transfection in hES and human embryonal carcinoma cell lines.
  • To identify promoters that support robust and sustained transgene expression in hES cells.
  • To evaluate the impact of specific promoter elements on gene silencing.

Main Methods:

  • Transient and stable transfection assays were performed in hES and embryonal carcinoma cell lines.
  • Enhanced green fluorescent protein (eGFP) was used as a reporter gene.
  • Four promoters were tested: UbiquitinC (pUbiC), Rosa26 (pR26), human cytomegalovirus major immediate early promoter (HCMV-MIE; pCMV), and a hybrid promoter (pCAGG).
  • The effect of the polyoma virus mutant enhancer PyF101 in the pCAG vector was also assessed.

Main Results:

  • Transient transfection results varied unpredictably across cell lines for all promoters.
  • Stable transfection showed significant transgene silencing, particularly with the pCMV promoter, yielding no eGFP-positive clones.
  • The pCAG vector, containing the CAGG promoter linked to the PyF101 enhancer, successfully generated stable eGFP-positive clones with long-term expression (>120 passages).
  • Removal of the PyF101 elements from the pCAG vector resulted in progressive gene silencing.
  • pUbiC, pR26, pCAGG, and pCAG demonstrated superiority over pCMV for stable hES cell transfections.

Conclusions:

  • Promoter choice is a critical determinant for achieving stable transgene expression in hES cells.
  • The pCAG promoter, when combined with the PyF101 enhancer, is highly effective for sustained gene expression in hES cells.
  • Mammalian promoters like pUbiC, pR26, and pCAGG are generally more suitable than pCMV for generating stable hES cell transfectants.