Transgenic mice with defined combinations of drug-inducible reprogramming factors (original) (raw)

Drug-inducible lentiviruses encoding Oct4, Sox2, Klf4 and c-Myc were used to derive "primary" iPS cells and segregated through germline transmission generating mice and fibroblasts carrying subsets of the reprogramming factors. Drug treatment of the cells resulted in "secondary" iPS cell derivation only when the missing factor was introduced. This creates a defined platform for studying reprogramming mechanisms and allows screening of genetically homogenous cells for compounds that replace any transcription factor required for iPS cell derivation. The generation of induced pluripotent stem (iPS) cells from mouse and human somatic cells through the forced expression of defined transcription factors1-4 constitutes a major breakthrough in regenerative biology5. However, current reprogramming strategies require viral transduction with potentially oncogenic transcription factors. Understanding the molecular changes underlying iPS cell derivation will be required to devise alternative and safer strategies for reprogramming e.g., by replacing the viral transduced factors with small molecules6-8. Screening approaches using infected cells are hampered by the genetic variability caused by the random integrations of multiple proviral copies9,10. Recently, we generated a "secondary" transgenic system that eliminates such heterogeneity9,10. In this approach, mouse embryonic fibroblasts (MEFs) heterozygous for the ROSA26-M2 reverse tetracycline transactivator (M2-rtTA) were infected with Doxycycline (Dox)-inducible lentiviruses carrying the four reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) and induced to generate "primary" iPS cells by addition of Dox. These cells were used to obtain chimeric mice with genetically identical somatic cells that can be isolated and reprogrammed in vitro by addition of Dox. However, such "secondary" somatic cells require isolation from Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: