A versatile bimodal QF/QS gene expression system for insect and vertebrate models based on conditional protein self-splicing

Summary

Re-engineering the Q-system for precise tissue- and temporally-specific gene expression manipulation in insect and vertebrate models by incorporating temperature-sensitive self-splicing intein modules.

What they want

The Q-system (QF2 driver and QUAS reporters) will be re-engineered to remedy limitations like toxicity and lack of an effective suppressor. This involves incorporating temperature-sensitive self-splicing intein modules (INTts) into the DNA binding domain of QF2 (QF2_INTts) and the protein interaction domain of QS (QS_INTts). These inteins will disrupt function at restrictive temperatures (24 to 30°C) and activate QF and QS via self-splicing at permissive temperatures (17 to 23°C). Transgenic Drosophila will be tested for functionality using QUAS-GFP reporter genes at different temperatures. Genes encoding validated QF2_INTts will be conferred to plasmids for Danio rerio transgenic fish and tested for functionality using QUAS-GFP reporters.

Deliverables

Re-engineered Q-system with temperature-sensitive self-splicing intein modules (QF2_INTts and QS_INTts)
Transgenic Drosophila tested for functionality of QF2_INTts and QS_INTts
Plasmids encoding validated QF2_INTts for D. rerio transgenic fish
Transgenic D. rerio fish tested for functionality of QF2_INTts

Technical requirements

Use of CRISPR/Cas-9 technology
Incorporation of temperature-sensitive self-splicing intein modules (INTts)
Permissive temperature setpoints between 17 and 23°C
Restrictive temperature setpoints between 24 and 30°C
Integration into the DNA binding domain of QF2 and the protein interaction domain of QS
Use of QUAS-GFP reporter genes