Song:Experimental Design For Brainbow
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Aim 1.To generate cell lines random-integrated with brainbow cassette
- To construct lentiviral vector carrying Brainbow-2.1 cassette
- To package and transfect lentivirus
- To isolate GFP-positive cells by flow cytometric cell sorting
- To establish integrated clonal cell line from single cell
- To validate the persistency and efficiency of GFP expression after passaging and during differentiation
Aim 2.To introduce inducible Cre into brainbow-integrated cell lines
- To construct inducible Cre with a drug-selection marker
- CAG-CreER regulated by tamoxifen
- Or tetO-Cre regulated by doxycycline
- To establish Cre-integrated stable cell lines by adding drug
- To validate stochastic fluorescence labeling after the addition of tamoxifen or doxycycline to the culture
Aim 3.To establish live cell imaging and automated lineage tracking system
Aim 4.To manually isolate single cell from differentiation culture
Aim 5.To perform single-cell transcriptomic and epigenomic analysis