title: Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells.
abstract: The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase. The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype, and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into 'safe harbor' sites in the genomes of autologous, patient-derived iPS cells.
authors.affiliation: Max Delbrück Center for Molecular Medicine, Robert Rössle Strasse 10, 13125 Berlin, Germany.
authors.forename: Ivana
authors.initials: I
authors.lastname: Grabundzija
authors.forename: Jichang
authors.initials: J
authors.lastname: Wang
authors.forename: Attila
authors.initials: A
authors.lastname: Sebe
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Erdei
authors.forename: Robert
authors.initials: R
authors.lastname: Kajdi
authors.forename: Anantharam
authors.initials: A
authors.lastname: Devaraj
authors.forename: Doris
authors.initials: D
authors.lastname: Steinemann
authors.forename: Károly
authors.initials: K
authors.lastname: Szuhai
authors.forename: Ulrike
authors.initials: U
authors.lastname: Stein
authors.forename: Tobias
authors.initials: T
authors.lastname: Cantz
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.forename: Christopher
authors.initials: C
authors.lastname: Baum
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Izsvák
authors.forename: Balázs
authors.initials: B
authors.lastname: Sarkadi
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Ivics
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D004251:DNA Transposable Elements; D055879:Gene Knock-In Techniques; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D019426:Integrases / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051379:Mice; D019895:Transposases / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; C045073:Cre recombinase; D019426:Integrases; D019895:Transposases; C494471:sleeping beauty transposase, human