RNA interference with FOXL2 and DMRT1

Highly-Experimental Research PLEASE DON'T TRY THIS AT HOME! This is highly experimental and incredibly dangerous stuff. Though if you are a professional looking for some new research idea, then by all means you're welcome to use our hypothesis in a study. ❤

This is a moon-shot project to reprogram the testes into ovaries, or the ovaries into testes! (Kinda.)

The war between FOXL2 (♀) and SOX9/DMRT1 (♂)

In the SRY signaling cascade, gonads have a sort of "toggle switch." If FOXL2 is active, then SOX9 is suppressed and the gonads become ovaries. If SOX9 is active, then FOXL2 is suppressed an the gonads become ovaries. Remarkably, researchers have reprogrammed the ovaries into testes by blocking FOXL2 in adult mice! Similarly, blocking DMRT1 in adult mice reprograms the testes into "mini-ovaries." Unfortunately, both variants were infertile, but we saw the cells reorganize, "cis" levels of hormones produced etc. So what if we could block these genes in adult humans?

RNA Interference

RNA interference is a technique to silence the transcription of selected genes, by producing an RNA strand complimentary to the mRNA transcript of the selected gene which will be recognized as belonging to an invading virus by the body, and blocked from getting to the ribosome. SNALPs are lipid membranes which contain copies of siRNA. When they hit a cell, they fuse with the cell membrane, disbursing the interfering RNA into the cell.

Proposal

We could use RNA interference to block either FOXL2 or DMRT1. Several drugs currently in phase III FDA trials use RNAi, which seems to demonstrate that in principle enough of the kinks have been worked out to try on humans. We'd find a region of messenger RNA for FOXL2 or DMRT1 that could be blocked using RNAi, then encase the interfering siRNA in SNALPs and deliver it to the cells, probably by injection or whatever the FDA trials did. We should be able to judge efficacy by trying it in testis/ovary tissue culture.

Synthesis Steps

• Identify interfering siRNA sequence
• Acquire DNA for siRNA
• RNA polymerase to amplify siRNA
• SNALP formation
• Sonication to embed siRNA within SNALP
• Safety checks on siRNA purity

Research Wishlist

• SNALP preparation protocol
• Protocols used by FDA trials of RNAi
• Previous roadblocks with RNAi

References

1. DMRT1 prevents female reprogramming in the postnatal mammalian testis
2. Sexual Cell-Fate Reprogramming in the Ovary by DMRT1 (supplementary)
3. Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation (open access!)
4. Sex reversal following deletion of a single distal enhancer of Sox9.