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Induced Pluripotent Stem Cells01:13

Induced Pluripotent Stem Cells

Stem cells are undifferentiated cells that divide and produce different types of cells. Ordinarily, cells that have differentiated into a specific cell type are post-mitotic—that is, they no longer divide. However, scientists have found a way to reprogram these mature cells so that they “de-differentiate” and return to an unspecialized, proliferative state. These cells are also pluripotent like embryonic stem cells—able to produce all cell types—and are therefore called induced pluripotent stem...

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Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation
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Creation of engineered human embryonic stem cell lines using phiC31 integrase.

Bhaskar Thyagarajan1, Ying Liu, Soojung Shin

  • 1Invitrogen Corporation, Carlsbad, California 92008, USA. bhaskar.thyagarajan@invitrogen.com

Stem Cells (Dayton, Ohio)
|October 27, 2007
PubMed
Summary

The phiC31 integrase efficiently targets pseudo-attachment sites in human embryonic stem cells (hESC). This method creates stable, engineered hESC clones that retain differentiation potential and specific gene expression.

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Area of Science:

  • Stem Cell Biology
  • Molecular Biology
  • Genetic Engineering

Background:

  • Phage-derived phiC31 integrase is known to target pseudo-attachment sites in various species.
  • Previous studies demonstrated its efficiency in cultured cells and in vivo.
  • The utility of phiC31 integrase in human embryonic stem cells (hESC) remained to be demonstrated.

Purpose of the Study:

  • To demonstrate the utility of phiC31 integrase for site-specific integration in hESC.
  • To create engineered hESC clones with stable expression of reporter genes.
  • To assess the differentiation potential and gene expression fidelity of engineered hESC.

Main Methods:

  • Generated hESC-derived clones using variant lines BG01v and SA002.
  • Introduced expression constructs for green fluorescent protein (GFP) under Oct4 or EF1alpha promoters.
  • Selected stable clones via antibiotic resistance and mapped integration sites using plasmid rescue.

Main Results:

  • Identified candidate genomic hot spots for integration in hESC, with a distinct pseudo-attP profile compared to differentiated cells.
  • Engineered hESC clones retained the ability to differentiate into all three germ layers.
  • GFP expression fidelity was confirmed in differentiation assays; Oct4 promoter recapitulated endogenous Oct4, while EF1alpha showed persistent expression.

Conclusions:

  • PhiC31 integrase is effective for targeting pseudo-attP sites in hESC.
  • Integrase-mediated site-specific integration efficiently generates stably expressing engineered hESC clones.
  • This technology facilitates the creation of valuable tools for stem cell research and therapeutic applications.