Pluripotentiality and Conditional Transgene Regulation In Human Embryonic Stem Cells Expressing Insulated Tet-on Transactivator

¹ Stem Cells and Regenerative Medicine Center (STaR), and Center for Cell and Gene Therapy (CAGT), Department of Pediatrics, Baylor College of Medicine

^* To whom correspondence should be addressed. E-mail: Goodell{at}bcm.edu.

	Abstract

Conditional manipulation of gene expression by using tetracycline (TET)-ON based approaches has proven invaluable to study fundamental aspects of biology; however, the functionality of these systems in human embryonic stem cells (hESC) has not been established. Given the sensitivity of these cells to both genetic manipulation and variations of culture conditions, constitutive expression of TET transactivators might not only be toxic for hESC but also impair their ability to self-renew or differentiate into multiple tissues. Therefore, the effect of these transactivators on the biology and pluripotentiality of hESC must first be evaluated before broad use of TET-ON methodologies is applied in these cells. Improved insulated lentivectors that display stable transgene expression and minimal insertional transactivation have been described for hESC. By using insulated lentivectors that allow simultaneous expression of TET components and fluorescent reporters, here we demonstrate that hESC constitutively expressing the TET-ON transactivator rtTA2SM2 can be derived and expanded in culture while retaining inducible transgene expression and pluripotentialty, including marker expression, a normal karyotype, and the ability to generate multiple tissues of different germ layer origin in teratomas. We also show that these cells retain the ability to control the expression of a stable integrated transgene in a dox-dependent manner, which demonstrates that an insulated TET-ON lentiviral system is functional in hESC. Together, our results indicate that improved TET regulators like rtTA2SM2 in combination with insulated lentiviral-based systems offer alternative strategies for conditional gene expression in hESC cells.

This article has been cited by other articles:

				X. Xia, M. Ayala, B. R. Thiede, and S.-C. Zhang In Vitro- and In Vivo-Induced Transgene Expression in Human Embryonic Stem Cells and Derivatives Stem Cells, February 1, 2008; 26(2): 525 - 533. [Abstract] [Full Text] [PDF]