Loss-of-function studies are powerful tool for understanding a gene function. Conditional gene silencing is a more favorable approach than gene knockout for studying essential genes’ functions due to the cytotoxicity of the latter. Another obstacle for analyzing essential genes is the need of tools that allow tight and rapid gene silencing. The lack of convenient methodologies to generate conditional gene silencing human cell lines prompted me to develop several methods for gene silencing. A set of Sleeping Beauty transposon vectors were developed based on a system that combined tetracycline-controlled promoter and auxin-inducible degron (tTA–AID), which enabled fast and robust gene silencing. When this system was concurrently utilized with CRISPR-Cas9 to disrupt the endogenous gene, c...[ Read more ]

Loss-of-function studies are powerful tool for understanding a gene function. Conditional gene silencing is a more favorable approach than gene knockout for studying essential genes’ functions due to the cytotoxicity of the latter. Another obstacle for analyzing essential genes is the need of tools that allow tight and rapid gene silencing. The lack of convenient methodologies to generate conditional gene silencing human cell lines prompted me to develop several methods for gene silencing. A set of Sleeping Beauty transposon vectors were developed based on a system that combined tetracycline-controlled promoter and auxin-inducible degron (tTA–AID), which enabled fast and robust gene silencing. When this system was concurrently utilized with CRISPR-Cas9 to disrupt the endogenous gene, conditional gene silencing cell lines could be generated using a one-step procedure. Cell lines with conditional silencing of multiple genes could also be generated simultaneously or sequentially. A shortcoming of AID is that portions of AID-tagged proteins could be degraded even in the absence of auxin. I showed the background degradation of AID-tagged proteins could be alleviated by using another F-box protein called AFB2. To achieve a more rigorous analysis of a gene’s functions, I further developed two new systems for conditional control of protein expression. In the first system, a rapid and tunable gene activation system was built based on a DB-EcR promoter and mutated FKBP12-derived destabilization domain degron.

When used in conjunction with the tTA–AID system to control a second gene, two genes could be rapidly and tightly switched around. The second system was based on a LacI-VP48 promoter and IKZF3 degron. The degradation efficiency was increased when tagging IKZF3 to both the target protein and LacI-VP48. When used together with the tTA–AID system, two genes could be controlled independently and degraded in a fast and complete manner. These resources are general molecular tools that will allow researchers to understand essential genes’ functions.