Song:Experimental Design For Brainbow: Difference between revisions

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<b>Aim 1.</b>To generate cell lines random-integrated with brainbow cassette
<b>Aim 1.</b> To generate cell lines random-integrated with brainbow cassette
*To construct lentiviral vector carrying Brainbow-2.1 cassette
*To construct lentiviral vector carrying Brainbow-2.1 cassette
*To package and transfect lentivirus
*Lentivirus package and transfection
*To isolate GFP-positive cells by flow cytometric cell sorting
*Isolating GFP-positive cells by flow cytometric cell sorting
*To establish integrated clonal cell line from single cell
*Establishing integrated clonal cell line from single cell
*To validate the persistency and efficiency of GFP expression after passaging and during differentiation
*Validating 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
*To construct inducible Cre with a drug-selection marker
**CAG-CreER regulated by tamoxifen
**CAG-CreER regulated by tamoxifen
**Or tetO-Cre regulated by doxycycline
**Or tetO-Cre regulated by doxycycline
*To establish Cre-integrated stable cell lines
*Establishing Cre-integrated stable cell lines by drug selecting
*To validate stochastic fluorescence labeling after adding drug to the culture
*Validating stochastic fluorescence labeling after the addition of tamoxifen or doxycycline 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
*Acquiring fluorescence images from all channel at a defined interval
*Processing acquired images and segmenting cells from the images
*To develop algorithms for automated lineage tracking


<b>Aim 4.</b>To manually isolate single cell from differentiation culture
<b>Aim 4.</b> To manually isolate single cell from differentiation culture
*Determining the time required for XFP expression and for cells to complete mitosis after drug addition
*Isolating single cells by local trypsination and micromanipulation
*Isolating the differentiation derivatives of the siblings of the above step
*Recording the spatial and color information of each isolated cells


<b>Aim 5.</b>To perform single-cell transcriptomic and epigenomic analysis
 
<b>Aim 5.</b> To perform single-cell transcriptomic and epigenomic analysis
*Single cell toto-RNAseq
*Characterizing DNA accessibility on limited cells by THS-seq

Latest revision as of 19:16, 6 September 2013

Aim 1. To generate cell lines random-integrated with brainbow cassette

  • To construct lentiviral vector carrying Brainbow-2.1 cassette
  • Lentivirus package and transfection
  • Isolating GFP-positive cells by flow cytometric cell sorting
  • Establishing integrated clonal cell line from single cell
  • Validating 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
  • Establishing Cre-integrated stable cell lines by drug selecting
  • Validating 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

  • Acquiring fluorescence images from all channel at a defined interval
  • Processing acquired images and segmenting cells from the images
  • To develop algorithms for automated lineage tracking


Aim 4. To manually isolate single cell from differentiation culture

  • Determining the time required for XFP expression and for cells to complete mitosis after drug addition
  • Isolating single cells by local trypsination and micromanipulation
  • Isolating the differentiation derivatives of the siblings of the above step
  • Recording the spatial and color information of each isolated cells


Aim 5. To perform single-cell transcriptomic and epigenomic analysis

  • Single cell toto-RNAseq
  • Characterizing DNA accessibility on limited cells by THS-seq