title: Pluripotentiality and conditional transgene regulation in human embryonic stem cells expressing insulated tetracycline-ON transactivator.
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 might 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 pluripotentiality, 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 doxycycline-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. Disclosure of potential conflicts of interest is found at the end of this article.
authors.affiliation: Stem Cells and Regenerative Medicine Center, Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, N1030, Houston, Texas 77030, USA.
authors.forename: Diego S
authors.initials: DS
authors.lastname: Vieyra
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Goodell
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D004318:Doxycycline / Q000494:pharmacology*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D038101:Insulator Elements / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013724:Teratoma / Q000473:pathology; D015533:Transcriptional Activation / Q000187:drug effects*; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C083139:Tet O resistance protein, Bacteria; C470187:TetR(B) protein, E coli; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly; D004318:Doxycycline