title: Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.
abstract: Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.
authors: Chair of Livestock Biotechnology, Technische Universität München, Freising, Germany.
authors: Claudia
authors: C
authors: Merkl
authors: Anja
authors: A
authors: Saalfrank
authors: Nathalie
authors: N
authors: Riesen
authors: Ralf
authors: R
authors: Kühn
authors: Anna
authors: A
authors: Pertek
authors: Stefan
authors: S
authors: Eser
authors: Markus Sebastian
authors: MS
authors: Hardt
authors: Alexander
authors: A
authors: Kind
authors: Dieter
authors: D
authors: Saur
authors: Wolfgang
authors: W
authors: Wurst
authors: Antonio
authors: A
authors: Iglesias
authors: Angelika
authors: A
authors: Schnieke
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D018929:Cell Culture Techniques; D054337:Cell Dedifferentiation / Q000235:genetics*; D002460:Cell Line; D058732:Embryoid Bodies / Q000502:physiology; D016259:Genes, myc / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000379:methods
chemical_list: C000715469:Klf4 protein, rat; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; D055748:SOXB1 Transcription Factors; C527784:Sox2 protein, rat
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