Project description:We have previously shown that pluripotent stem cells are induced from mouse fibroblasts by retroviral introduction of Oct3/4, Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 expression. These iPS (induced pluripotent stem) cells are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation. Fbx15-iPS cells, however, are different in gene expression and DNA methylation patterns and fail to produce adult chimeras. Here we show that selection for Nanog results in germ-line competent iPS cells, with more ES cell-like gene expression and DNA methylation patterns. The four transgenes were strongly silenced. We obtained adult chimeras from seven Nanog-iPS clones, with one clone transmitted through the germ-line to the next generation. Approximately 20% of the offspring developed tumors, where c-Myc transgene was reactivated. Thus iPS cells competent for germ-line chimeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application. Keywords: cell type comparison
Project description:We have previously shown that pluripotent stem cells are induced from mouse fibroblasts by retroviral introduction of Oct3/4, Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 expression. These iPS (induced pluripotent stem) cells are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation. Fbx15-iPS cells, however, are different in gene expression and DNA methylation patterns and fail to produce adult chimeras. Here we show that selection for Nanog results in germ-line competent iPS cells, with more ES cell-like gene expression and DNA methylation patterns. The four transgenes were strongly silenced. We obtained adult chimeras from seven Nanog-iPS clones, with one clone transmitted through the germ-line to the next generation. Approximately 20% of the offspring developed tumors, where c-Myc transgene was reactivated. Thus iPS cells competent for germ-line chimeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application. Experiment Overall Design: Total RNA from Fbx15-null MEF (duplicate), Fbx15-null ES cells (duplicate), Fbx15-iPS cells (clone MEF4-7, duplicate), or Nanog-iPS cells (clones 20D -2, 16, 17, and 18) were labeled with Cy3. Samples were hybridized to a Mouse Oligo Microarray (Agilent) according to the manufacturerâs protocol. Arrays were scanned with a G2565BA Microarray Scanner System (Agilent). Data were analyzed using GeneSprings GX software (Agilent). We excluded genes whose value fluctuated more than 2-fold between duplicated analyses.
Project description:We developed and report on a novel new technique to reprogramm fobroblasts from two non-permissive mouse backgrounds into emrbyonic stem cell-like induced pluripotent stem cells
