title: Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system.
abstract: One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo.
authors.affiliation: Molecular Animal Biotechnology Laboratory, Szent István University, 2100 Gödöllő, Hungary; BioTalentum Ltd., Aulich Lajos Street 26, 2100 Gödöllő, Hungary.
authors.forename: E
authors.initials: E
authors.lastname: Varga
authors.affiliation: BioTalentum Ltd., Aulich Lajos Street 26, 2100 Gödöllő, Hungary.
authors.forename: C
authors.initials: C
authors.lastname: Nemes
authors.affiliation: Department of Anatomy & Embryology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.
authors.forename: R P
authors.initials: RP
authors.lastname: Davis
authors.affiliation: BioTalentum Ltd., Aulich Lajos Street 26, 2100 Gödöllő, Hungary.
authors.forename: O
authors.initials: O
authors.lastname: Ujhelly
authors.affiliation: BioTalentum Ltd., Aulich Lajos Street 26, 2100 Gödöllő, Hungary; Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, 10700 Bangkok, Thailand.
authors.forename: N
authors.initials: N
authors.lastname: Klincumhom
authors.affiliation: BioTalentum Ltd., Aulich Lajos Street 26, 2100 Gödöllő, Hungary.
authors.forename: Z
authors.initials: Z
authors.lastname: Polgar
authors.affiliation: BioTalentum Ltd., Aulich Lajos Street 26, 2100 Gödöllő, Hungary; Department of Farm Animal Health and Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands.
authors.forename: S
authors.initials: S
authors.lastname: Muenthaisong
authors.affiliation: BioTalentum Ltd., Aulich Lajos Street 26, 2100 Gödöllő, Hungary.
authors.forename: M K
authors.initials: MK
authors.lastname: Pirity
authors.affiliation: Molecular Animal Biotechnology Laboratory, Szent István University, 2100 Gödöllő, Hungary; BioTalentum Ltd., Aulich Lajos Street 26, 2100 Gödöllő, Hungary; Department of Farm Animal Health and Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands. Electronic address: andras.dinnyes@biotalentum.hu.
authors.forename: A
authors.initials: A
authors.lastname: Dinnyes
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015139:Blotting, Southern; D015153:Blotting, Western; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D018274:Electroporation; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors; D057809:HEK293 Cells; D006321:Heart / Q000196:embryology / Q000502:physiology; D006801:Humans; D007124:Immunoenzyme Techniques; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D019426:Integrases / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D012333:RNA, Messenger; C045073:Cre recombinase; D019426:Integrases; D000469:Alkaline Phosphatase
keywords: Differentiation; Germ-line; Mouse; Pluripotent; Reprogramming; Stem cell