As some of you might know, we have teamed up with Gabriel Licina and his crew for this experiment. Recently, we finalized the design of our custom combined vector carrying the rtTA gene
and the Oct4 gene under the Tet-On system
. Here is our final map:
Once the vector build is complete, we will check that it works by transfecting human dermal fibroblasts and measuring Oct4 expression levels with and without Dox.
After that, we will carry out the main experimental procedures of differential Dox induction in human dermal fibroblasts (from a donor aged 40-50) transfected with our vector, collect resulting cells and ship them off to Zymo for methylation clock analysis.
Our intended dosing protocol is as follows: induce Oct4 expression in transfected HDF for 1,2,3 or 5 consecutive days via Dox. On the final day of each induction cycle, harvest part of the culture to be sent for sequencing. Use the remaining part of the culture for relaxation/washout experiments to see if methylation returns to its original configuration: i.e. stop Dox induction and harvest cells after 2,6,12 days after Dox withdrawal.
Stay tuned for further updates!