Song:Experimental Design For Protein Tagging

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Aim 1. To generate cell lines integrated with CD-tagging vector pBabeAE

  • Retrovirus package and transfection
  • Establishing stable cell lines by puromycin selection


Aim 2. To isolate cells expressing fluorescence at various intermediate stages by FACS

  • Differentiating integrated cell lines into any desired cell type
  • To isolate cells expressing fluorescence by FACS


Aim 3. To revert isolated differentiated cells to pluripotency

  • Treating isolated cells by doxycycline to induce reprogramming
  • Isolating successfully reprogrammed iPS cells by cell sorting using ES specific surface makers such as TRA-1-60
  • Culturing collect cells as an unique cell library for proteins expressed at any interesting time point


Aim 4. To characterize insertion sites and subclone each cells

  • Differentiating cultured cell library into the stage when cells were original isolated
  • Pool RNA-seq to identify all the insertion sites in the library
  • Subcloning cell lines carrying proteins with a temporal expression pattern of interested from single cells