Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon

Area of Science:

• * Molecular Biology
• * Genetics
• * Transposon Technology

Background:

• * Insertional mutagenesis using transposable elements is a key tool in model organisms.
• * The Sleeping Beauty (SB) transposon is an effective insertional mutagen in mouse germline studies.
• * Existing SB transposon vectors have limitations in reporting gene expression and enabling conditional gene expression.

Purpose of the Study:

• * To develop a Sleeping Beauty (SB) transposon system for reporting endogenous mouse gene expression.
• * To create a system for conditional gene expression in a tissue- or temporal-specific manner.
• * To integrate the doxycycline-repressible Tet-Off (tTA) system for regulated gene activation.

Main Methods:

• * Systematic development and testing of a transposon-based gene-trap system incorporating the Tet-Off (tTA) system.
• * In vitro validation using human cells and transposition of the "gene-trap tTA" vector.
• * In vivo testing in transgenic mice, including assessment of germline mobilization rates and gene insertion analysis.

Main Results:

• * The gene-trap tTA system demonstrated full functionality in vitro, activating TRE-luciferase transgene expression in a doxycycline-dependent manner.
• * In vivo studies confirmed germline mobilization of gene-trap tTA vectors and identified candidate gene insertions.
• * The gene-trap system successfully reported endogenous gene expression and was coupled with bioluminescent imaging for tissue-specific expression analysis.

Conclusions:

• * The developed gene-trap tTA vector system functions similarly to the GAL4/UAS system in flies for mouse gene analysis.
• * This tool facilitates both the annotation of mouse genes and the creation of resources for conditional gene expression studies.
• * The system provides a valuable resource for the mouse genetics community to dissect mammalian gene function through conditional expression and mutagenesis.