Abstract
SummaryBinary gene regulatory tools such as the Tetracycline (Tet)-controlled transcription system have revolutionized genetic research in multiple organisms, but their applications to the worm remain very limited. Here we report that the canonical Tet system is largely inactive in the worm but can be adapted for the worm by introducing multiple modifications, a crucial one being the use of the transcription activation domain from the fungal Q binary system. The resultant Tet/Q hybrid system proves more robust and flexible than either of its precursors, enabling elaborate modes of transgene manipulation previously hard to achieve in the worm, including inducible intersectional regulation and, in combination with the Q system, independent control of distinct transgenes within the same cells. Furthermore, we demonstrated, as an example of its applications, that the hybrid system can tightly and efficiently control Cre expression. This study establishes Tet/Q as a premier binary system for worm genetic research.
Highlights
Genetic tools for controlling transcription of engineered transgenes have revolutionized biomedical research and biotechnology
Adaption of the Tet System for the Worm Our preliminary data indicate that the canonical Tet system is largely inactive in the worm
We found that the modified Tet system remained inefficient even when we replaced the VP16 activation domain in rtTA with VP64, the tetramerized minimal VP16 activation domain (Beerli et al, 1998) or VPR, the super-strong, tripartite activation domain comprising VP16 and two other mammalian activator domains (Chavez et al, 2015) (Figure 2A)
Summary
Genetic tools for controlling transcription of engineered transgenes have revolutionized biomedical research and biotechnology Among such tools, one of the most powerful is the Tetracycline (Tet) regulatory system, which allows efficient, graded, reversible, and spatiotemporal transcription regulation by Tet (Berens and Hillen, 2003; Schonig et al, 2010) (Figure 1A). The two eukaryotic components of the Tet system, the VP16 activation domain and the CMV minimal promoter (Figure 1A), are highly effective. Tetracyclines and their analog Doxycycline (Dox) can readily permeate tissues and are well tolerated, and their pharmacology has been thoroughly analyzed because of their medical importance. Applications to the nematode C. elegans, a crucial model organism, are conspicuously absent
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