Song:Experimental Design For Brainbow: Difference between revisions

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*To isolate GFP-positive cells by flow cytometric cell sorting
*To isolate GFP-positive cells by flow cytometric cell sorting
*To establish integrated clonal cell line from single cell
*To establish integrated clonal cell line from single cell
*To validate persistent and efficient expression of GFP after passaging and during differentiation
*To validate the persistency and efficiency of GFP expression after passaging and during differentiation




<b>Aim 2.</b>To introduce inducible Cre into brainbow-integrated cell lines
<b>Aim 2.</b>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
*To validate stochastic fluorescence labeling after adding drug to the culture


<b>Aim 3.</b>To establish live cell imaging and automated lineage tracking system
<b>Aim 3.</b>To establish live cell imaging and automated lineage tracking system

Revision as of 17:38, 6 September 2013

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
  • To validate stochastic fluorescence labeling after adding drug 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