Generation of mouse induced pluripotent stem cells from different genetic backgrounds using Sleeping beauty transposon mediated gene transfer

Induced pluripotent stem (iPS) cell technology involves reprogramming somatic cells to a pluripotent state. The original technology used to produce these cells requires viral gene transduction and results in the permanent integration of exogenous genes into the genome. This can lead to the development of abnormalities in the derived iPS cells. Here, we report that non-viral transfection of a Sleeping Beauty (SB) transposon containing the coding sequences Oct3/4 (Pouf1), Sox-2, Klf-4 and c-Myc (OSKM) linked with 2A peptides, can reprogram mouse fibroblasts. We have established reprogrammed mouse cell lines from three different genetic backgrounds: (1) ICR - outbred, (2) C57BL/6 - inbred and (3) F1-hybrid (C57BL/6 x DBA/2J), with parallel robust expression of all exogenous (Oct3/4, Sox-2, Klf-4, and c-Myc) and endogenous (e.g. Oct3/4 and Nanog) pluripotency genes. The iPS cell lines exhibited characteristics typical for undifferentiated embryonic stem (ES) cell lines: ES cell-like morphology, alkaline phosphatase (ALP) positivity and gene expression pattern (shown by reverse transcription PCR, and immunofluorescence of ES cell markers - e.g. Oct3/4, SSEA1, Nanog). Furthermore, cells were able to form embryoid bodies (EBs), to beat rhythmically, and express cardiac (assayed by immunofluorescence, e.g. cardiac Troponin T, desmin) and neuronal (assayed by immunofluorescence e.g. nestin, Tuj1) markers. The in vitro differentiation potential was found to be the highest in the ICR-derived iPS lines (ICR-iPS). Interestingly, the ICR-iPS lines had even higher differentiation potential than the ICR-ES cell lines: the rate of EBs forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. In vivo, the ICR and F1 hybrid iPS cells formed chimeras and one of the iPS cells from the F1 hybrid background transmitted to the germline. Our results suggest that iPS technology may be useful for generating pluripotent stem cells from genetic backgrounds of which good quality ES cell generation is difficult. These studies provide insights into viral-free iPS technology and may contribute towards defining future cell-based therapies, drug-screening methods and production of transgenic animals using genetically modified iPS cells

Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system

Stem cells & developmental biolog

Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system

Stem cells & developmental biolog