Applying a "double-feature" promoter to identify cardiomyocytes differentiated from human embryonic stem cells following transposon-based gene delivery.
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.
Orbán
Tamás I
TI
Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, Budapest, Hungary.
Apáti
Agota
A
Németh
Andrea
A
Varga
Nóra
N
Krizsik
Virág
V
Schamberger
Anita
A
Szebényi
Kornélia
K
Erdei
Zsuzsa
Z
Várady
György
G
Karászi
Eva
E
Homolya
László
L
Német
Katalin
K
Gócza
Elen
E
Miskey
Csaba
C
Mátés
Lajos
L
Ivics
Zoltán
Z
Izsvák
Zsuzsanna
Z
Sarkadi
Balázs
B
D000818:Animals; D001483:Base Sequence; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002999:Clone Cells; D019295:Computational Biology; D004251:DNA Transposable Elements / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000166:cytology*; D018628:Gene Dosage; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D019076:Transgenes
D015415:Biomarkers; D004251:DNA Transposable Elements