Embryonic Stem Cells/Induced Pluripotent Stem Cells
Applying a "Double-Feature" Promoter To Identify Cardiomyocytes Differentiated From Human Embryonic Stem Cells Following Transposon-Based Gene Delivery
Tamás I. Orbán1§, Ágota Apáti1§, Andrea Németh1, Nóra Varga1, Virág Krizsik1, Anita Schamberger1, Kornélia Szebényi1, Zsuzsa Erdei1, György Várady1, Éva Karászi1, László Homolya1, Katalin Német1, Elen Gócza2, Csaba Miskey3, Lajos Mátés3, Zoltán Ivics3, Zsuzsanna Izsvák3, Balázs Sarkadi1*¶
1Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, Budapest, Hungary
2Genetic Modification Program Group, Agricultural Biotechnology Center, Gödöll, Hungary
3Mobile DNA Group, Max-Delbrück Center for Molecular Medicine, Berlin, Germany
email: Balázs Sarkadi (firstname.lastname@example.org)
*Correspondence to Balázs Sarkadi, Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, Dioszegi u. 64., Budapest, H-1113, Hungary
Author contributions: T.I.O.: Conception and design; collection and assembly of data; data analysis and interpretation; manuscript writing; Á.A.: Conception and design; collection and assembly of data; data analysis and interpretation; manuscript writing; A.N.: Collection and assembly of data; N.V.: Collection and assembly of data; V.K.: Collection and assembly of data; A.S.: Collection and assembly of data; K.S.: Collection and assembly of data; Z.E.: Collection and assembly of data; G.V.: Collection and interpretation of data; É.K.: Collection of data; L.H.: Interpretation of data; K.N.: Collection and interpretation of data; E.G.: Interpretation of data; C.M.: Data analysis; L.M.: Interpretation of data; Z.Ivics Data analysis; manuscript writing; Z.Izsvák: Data analysis and interpretation of data; B.S.: Conception and design; financial support; collection and assembly of data; data analysis and interpretation; manuscript writing; final approval of manuscript.
First published online in STEM CELLS Express February 20, 2009.
§Tamás I. Orbán and Ágota Apáti contributed equally to this work.
¶Phone/Fax: +36 1 372 4353
- EU FP6-INTHER; Grant Number: LSHB-CT-2005018961
- OTKA; Grant Number: AT 048986, NK72057, NKFP-1A-060/2004, ETT 405/2006
- KKK grants
Sleeping Beauty transposon • human embryonic stem cells • CAG promoter • double-feature promoter • cardiomyocytes • lentiviral gene delivery
Human embryonic stem (HuES) cells represent a new potential tool for cell- 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, 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, EF1, or PGK), 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 non-typical behavior of a constitutively active promoter, thereby avoiding invasive drug selection methods.
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