Conversion of fibroblasts to retinal cells by transcription (final)

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A presentation introducing my research project on the conversion of fibroblasts to retinal cells. This presentation summarizes my plans for the project.

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Conversion of fibroblasts to retinal cells by transcription (final)

  1. 1. Lucas Man Xiang Lab
  2. 2. <ul><li>The light-sensing tissue in eye </li></ul><ul><li>Consists of specialized neurons (photoreceptor, bipolar, horizontal, amacrine, and ganglion cells) and Müller glial cells arranged into layers </li></ul>Photoreceptors Bipolar & Horizontal Cells Amacrine Cells Ganglion Cells
  3. 3. <ul><li>Understanding retinal development </li></ul>Neuron 43:795-807 (2004)
  4. 4. <ul><li>Potential medical uses </li></ul><ul><ul><li>Generating functioning retinal cell types to treat congenital retinal disorders </li></ul></ul><ul><ul><li>Regenerating functioning retinal cell types to treat retinal disorders resulting from dead or impaired retinal cells </li></ul></ul>Retinitis pigmentosa Macular dystrophy
  5. 5. <ul><li>We are starting with fibroblasts, pluripotent cells whose fates can be reprogrammed by introducing transcription factors </li></ul><ul><li>We hope that by activating retinal-lineage-specific transcription factors in mouse embryonic fibroblasts (MEFs), we can induce the MEFs to differentiate into retinal cell types </li></ul>Scienceblogs Thermo Fisher Scientific, Inc.
  6. 6. <ul><li>Functional neurons have already been successfully created by conversion from fibroblasts through expression of a combination of only 3 transcription factors: Ascl1, Brn2, and Myt1l </li></ul><ul><ul><li>Thomas Vierbuchen et al. “Direct conversion of fibroblasts to functional neurons by defined factors”. Nature 469:1035-1041 (2010). </li></ul></ul><ul><li>Evidence that methodology is valid, the question is finding the right combination of transcription factors </li></ul>Nature 469:1035-1041 (2010)
  7. 7. <ul><li>17 factors specific to retinal cell lineages were selected: </li></ul><ul><ul><li>Pax6 </li></ul></ul><ul><ul><li>Rax/Rx </li></ul></ul><ul><ul><li>Sox2 </li></ul></ul><ul><ul><li>Six3/6 </li></ul></ul><ul><ul><li>Meis1/2 </li></ul></ul><ul><ul><li>Tlx/Nr2e1 </li></ul></ul><ul><ul><li>Otx2 </li></ul></ul><ul><ul><li>Crx </li></ul></ul><ul><ul><li>Nrl </li></ul></ul><ul><ul><li>Prdm1/Blimp1 </li></ul></ul><ul><ul><li>Math5 </li></ul></ul><ul><ul><li>Brn3b </li></ul></ul><ul><ul><li>Isl1 </li></ul></ul><ul><ul><li>Foxn4 </li></ul></ul><ul><ul><li>Math3 </li></ul></ul><ul><ul><li>Neurod1 </li></ul></ul><ul><ul><li>Ptf1a </li></ul></ul><ul><li>The 3 factors used in the Vierbuchen experiment from Nature will also be used as positive control </li></ul><ul><ul><li>Ascl1 </li></ul></ul><ul><ul><li>Brn2 </li></ul></ul><ul><ul><li>Myt1l </li></ul></ul>
  8. 8. <ul><li>Lentivirus-mediated transduction </li></ul>1. Clone factor by inserting factor of interest into cloning vectors 2. Sub-clone factor from cloning vector into virus vector 3. Transfect packaging cells with the viral vector and packaging plasmids 4. Harvest virus particles 5. Create viral cocktails and infect target cells 6. Stain to observe gene expression and identify presence of retinal-cell-specific markers
  9. 9. <ul><li>We hope to be able to induce MEFs to differentiate into a few retinal cell types by introducing transcription factors </li></ul><ul><li>Using lentivirus-mediated transduction, we will deliver the transcription factors of interest into MEFs </li></ul><ul><li>We hope to uncover new retinal cell development pathways and the functions of certain transcription factors, and perhaps open the door for future medical treatments </li></ul>
  10. 10. <ul><li>I’d like to thank: </li></ul><ul><li>my mentor Min Zou </li></ul><ul><li>the other graduate students and post-docs in the Xiang Lab </li></ul><ul><li>Dr. Mengqing Xiang </li></ul><ul><li>… And our mice ! </li></ul>

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