ABSTRACT
Reprogrammable mouse models engineered to conditionally express Oct-4, Klf-4, Sox-2 and c-Myc (OKSM) have been instrumental in dissecting molecular events underpinning the generation of induced pluripotent stem cells. However, until now these models have been reported in the context of the m2 reverse tetracycline-controlled transactivator, which results in low reprogramming efficiency and consequently limits the number of reprogramming intermediates that can be isolated for downstream profiling. Here, we describe an improved OKSM mouse model in the context of the reverse tetracycline-controlled transactivator 3 with enhanced reprogramming efficiency (>9-fold) and increased numbers of reprogramming intermediate cells albeit with similar kinetics, which we believe will facilitate mechanistic studies of the reprogramming process.
Subject(s)
Cellular Reprogramming , Tetracyclines/pharmacology , Transcriptional Activation/drug effects , Animals , Cell Differentiation/drug effects , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Mice , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Plasmids/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Teratoma/pathologyABSTRACT
The mammalian RFamide-related peptide RFRP1 was found to signal through the neuropeptide FF 2 receptor expressed in Xenopus oocytes. The peptide induced a dose-dependent outward current, which was dependent on the simultaneous expression of GIRK1 and GIRK4 potassium channels. In neuropathic rats, RFRP1 administered intrathecally induced tactile antiallodynia and thermal antinociception, whereas in the solitary tract nucleus it produced only mechanical antihyperalgesia. Expression of the RFamide-related peptide mRNA in the rat CNS was distinctly different from that of neuropeptide FF. Most notably, the gene was not expressed in the hindbrain or spinal cord at detectable levels. However, there was a prominent group of RFamide-related peptide mRNA-expressing neurons in the central hypothalamus, in the area in and between the dorsomedial and ventromedial nuclei. The results suggest that RFamide-related peptides are potentially involved in pain regulation through a hypothalamo-medullary projection system, and possibly via action on neuropeptide FF 2 receptors. In neuropathic animals, the pain suppressive effect of RFamide-related peptide varies depending on the submodality of noxious test stimulation and the site of RFamide-related peptide administration.
