Reprogramming Somatic Nuclei and Cells with Cell Extracts

Publisher Summary This chapter examines procedures for reprogramming somatic nuclei and cells with cell extracts. About 293T human fibroblasts are cultured on glass coverslips. Resuspend the cells in 10 ml ice-cold cell lysis buffer (CLB). It is preferable to use a graduated 15-ml conical tube to estimate the cell volume after sedimentation. Once lysis is achieved in a tube, keep the tube on ice and proceed with all other tubes. Power and duration of sonication vary with each cell type. In order for components from the reprogramming extract to enter 293T cells, the cells must be reversibly permeabilized. Permeabilization is accomplished with the Streptococcus pyogenes toxin, streptolysin O. SLO is a cholesterol-binding toxin that forms large pores in the plasma membrane of mammalian cells. Two additional coverslips should also be used as controls for the absence of SLO. Dilute the SLO stock in ice-cold HBSS to 230 ng/ml. Expression of new proteins can also be monitored at regular intervals after the reprogramming reaction by immunofluorescence or flow cytometry using standard protocols.