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Applying a "double-feature" promoter to identify cardiomyocytes differentiated from human embryonic stem cells following transposon-based gene delivery

Item Type:Article
Title:Applying a "double-feature" promoter to identify cardiomyocytes differentiated from human embryonic stem cells following transposon-based gene delivery
Creators Name:Orban, T.I. and Apati, A. and Nemeth, A. and Varga, N. and Krizsik, V. and Schamberger, A. and Szebenyi, K. and Erdei, Z. and Varady, G. and Karaszi, E. and Homolya, L. and Nemet, K. and Gocza, E. and Miskey, C. and Mates, L. and Ivics, Z. and Izsvak, Z. and Sarkadi, B.
Abstract:Human embryonic stem (HuES) cells represent a new potential tool for cell-therapy and gene-therapy applications. However, these approaches require the development of efficient, stable gene delivery, and proper progenitor cell and tissue separation methods. In HuES cell lines, we have generated stable, enhanced green fluorescent protein (EGFP)-expressing clones using a transposon-based (Sleeping Beauty) system. This method yielded high percentage of transgene integration and expression. Similarly to a lentiviral expression system, both the undifferentiated state and the differentiation pattern of the HuES cells were preserved. By using the CAG promoter, in contrast to several other constitutive promoter sequences (such as CMV, elongation factor 1alpha, or phosphoglycerate kinase), an exceptionally high EGFP expression was observed in differentiated cardiomyocytes. This phenomenon was independent of the transgene sequence, methods of gene delivery, copy number, and the integration sites. This "double-feature" promoter behavior, that is providing a selectable marker for transgene expressing undifferentiated stem cells, and also specifically labeling differentiated cardiomyocytes, was assessed by transcriptional profiling. We found a positive correlation between CAG promoter-driven EGFP transcription and expression of cardiomyocyte-specific genes. Our experiments indicate an efficient applicability of transposon-based gene delivery into HuES cells and provide a novel approach to identify differentiated tissues by exploiting a nontypical behavior of a constitutively active promoter, thereby avoiding invasive drug selection methods. Stem Cells 2009;27:1077-1087.
Keywords:Sleeping Beauty Transposon , Human Embryonic Stem Cells , CAG Promoter, Double-Feature Promoter, Cardiomyocytes, Lentiviral Gene Delivery, Animals, Mice
Source:Stem Cells
Publisher:AlphaMed Press
Page Range:1077-1087
Date:20 February 2009
Official Publication:https://doi.org/10.1002/stem.45
PubMed:View item in PubMed

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