Controlling Pancreas Cell Fate Using A Synthetic Actuator
- Controlling pancreas cell fate using transcription factors. Cell fate is defined as switching alpha (α) pancreas cells into beta (β) cells, the difference between the two is secretion of glucagon (α) and insulin (β).
- Pc-TF is a synthetically created fusion protein. The protein is a fusion of a transcription activation domain with a Polycomb chromodomain taken from PRC1 (H3K27me3 repressive complex). The binding of Pc-TF to H3K27me3 opens the gene and upregulates transcription of the gene. This upregulates the previously silenced gene. In pancreas cells, this would have the effect of switching cell fate from α to β. Αlpha cells are seen as undifferentiated cells, making it very possible for the cells states to be switched with this upregulation of gene targets repressed by H3K27me3 PRC1 binding.
- Galaxy, USCS Browser based bioinformatics to determine H32K27me3 methylation marks.
- Grow TC-1 Alpha Cells
- Transfect cells with Pc-TF expressing plasmid
- Incubate for 10 days (time points taken at 3,6,10 days)
- Check cells with flow cytology to confirm RFP expression (i.e. PcTF expression in cells)
- Extract mRNA from cells to create cDNA
- RT-PCR to determine gene expression through mRNA translation
- Cytology to confirm increase insulin/decrease glucagon