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| Item Type: | Article |
|---|---|
| Title: | Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector |
| Creators Name: | Merkl, C. and Saalfrank, A. and Riesen, N. and Kühn, R. and Pertek, A. and Eser, S. and Hardt, M.S. and Kind, A. and Saur, D. and Wurst, W. and Iglesias, A. and Schnieke, A. |
| 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 (Phi)C31 attB site and used a (Phi)C31 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. |
| Keywords: | Cell Culture Techniques, Cell Dedifferentiation, Cell Line, DNA Sequence Analysis, Embryoid Bodies, Genetic Vectors, Immunohistochemistry, Induced Pluripotent Stem Cells, Kruppel-Like Transcription Factors, Myc Genes, Octamer Transcription Factor-3, Reverse Transcriptase Polymerase Chain Reaction, Southern Blotting, SOXB1 Transcription Factors, Transfection, Animals, Rats |
| Source: | PLoS ONE |
| ISSN: | 1932-6203 |
| Publisher: | Public Library of Science |
| Volume: | 8 |
| Number: | 1 |
| Page Range: | e55170 |
| Date: | 31 January 2013 |
| Official Publication: | https://doi.org/10.1371/journal.pone.0055170 |
| PubMed: | View item in PubMed |
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