Song:Experimental Design For Protein Tagging
Jump to navigation
Jump to search
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