We plan to establish proof of concept for the ideal protein levels of recombinant proteins by attaching our synthetic genetic regulatory regions to red fluorescent protein (RFP) reporter, which can easily be quantified at a relatively low cost using an RFP quantification kit. We will then test protein levels in the same environments that erythroblasts might experience to insure that protein level change proportionally to the cell's stage of differentiation. Cells will be tested in multiple iron and heme conditions. After finding the regulatory elements which deliver the ideal levels of transcription, the process can be repeated but by attaching the selected regulatory elements the actual, erythropoiesis related genes.
The largest theoretical challenge will be choosing genetic regions which can provide the correct levels of gene expression through the entire process of erythropoiesis. The cis-acting elements in even the simplest mammalian promoters can be difficult to predict. So, even with transcription level estimates from a comprehensive literature review, we will still need to test multiple regulatory regions.
Our greatest experimental challenge will likely be reaching a high enough transfection efficiency by electroporation. Bone marrow, CD34+ cells are notoriously difficult to transfect with chemical methods, and viral vectors are vary expensive. We plan to optimize the electroporation protocol by a comprehensive literature review and conduct multiple trials.
We plan to use CD34+ bone marrow stem cells, but we are not going to actually induce differentiation until we have confirmed that the regulatory elements we have chosen will provide the correct protein levels. Using RFP as a model, we can quantitatively measure fluorescence with an RFP assay to determine the effect of our synthetic genetic regulatory regions at specific genetic loci. RFP levels can then be compared to ideal protein levels to determine in transcription needs to be up or down regulated.
Browse the protocols that are part of the experimental methods.