Abstract:
Regulatory T cells (Tregs) are an important part of the adaptive immune system. They function as negative regulators of the immune response, thereby preventing inflammatory diseases. They also maintain immunologic tolerance to self and commensal antigens. FOXP3, a transcription factor characterised as the master regulator of Tregs, can be modified in a variety of ways including epigenetic mechanisms. The FOXP3 locus contains a Treg specific demethylated region (TSDR) which is actively demethylated by Ten eleven translocation (TET) enzymes. This is important for the stability of FOXP3 expression and Treg development. Therefore, it is necessary to study them further to understand the underlying mechanisms. We sought to do this by using a TET1/TET2/TET3 triple knockdown approach to see the effect on development and functionality of in vitro induced Treg cells (iTregs) and further study their downstream targets. Firstly, we used a siRNA mediated approach which did not generate consistent results. Therefore we changed strategy and used a CRISPR/Cas9-based approach to stably silence the TET genes. We were successful in obtaining one functional CRIPSR crRNA for each TET which could efficiently knockdown the targeted gene for up to two weeks. Further, the procedure will have to be standardised for a triple knockdown.
