title: A robust strategy for negative selection of Cre-loxP recombination-based excision of transgenes in induced pluripotent stem cells.
abstract: Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells (iPSCs). For translation, it is important to silence or remove the reprogramming factors after induction of pluripotency. In this study, we design an excisable loxP-flanked lentiviral construct that a) includes all the reprogramming elements in a single lentiviral vector expressed by a strong EF-1α promoter; b) enables easy determination of lentiviral titer; c) enables transgene removal and cell enrichment using LoxP-site-specific Cre-recombinase excision and Herpes Simplex Virus-thymidine kinase/ganciclovir (HSV-tk/gan) negative selection; and d) allows for transgene excision in a colony format. A reprogramming efficiency comparable to that reported in the literature without boosting molecules can be consistently obtained. To further demonstrate the utility of this Cre-loxP/HSV-tk/gan strategy, we incorporate a non-viral therapeutic transgene (human blood coagulation Factor IX) in the iPSCs, whose expression can be controlled by a temporal pulse of Cre recombinase. The robustness of this platform enables the implementation of an efficacious and cost-effective protocol for iPSC generation and their subsequent transgenesis for downstream studies.
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.
authors.forename: Syandan
authors.initials: S
authors.lastname: Chakraborty
authors.forename: Nicolas
authors.initials: N
authors.lastname: Christoforou
authors.forename: Ali
authors.initials: A
authors.lastname: Fattahi
authors.forename: Roland W
authors.initials: RW
authors.lastname: Herzog
authors.forename: Kam W
authors.initials: KW
authors.lastname: Leong
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D015774:Ganciclovir / Q000494:pharmacology; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D019426:Integrases / Q000378:metabolism*; D051379:Mice; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics; D019076:Transgenes
chemical_list: D000998:Antiviral Agents; D017931:DNA Primers; D013937:Thymidine Kinase; C045073:Cre recombinase; D019426:Integrases; D015774:Ganciclovir