The piggyBac-based double-inducible binary vector system: A novel universal platform for studying gene functions and...

Area of Science:

• Molecular Biology
• Genetics
• Gene Expression Systems

Background:

• Eukaryotic inducible overexpression systems like Tet-On and mifepristone-inducible systems are crucial for reverse genetics studies.
• The piggyBac transposon system is highly efficient in cultured mammalian cells.
• Existing systems have limitations that necessitate improved tools for gene function analysis.

Purpose of the Study:

• To develop a novel piggyBac-based double-inducible system combining the strengths of Tet-On and mifepristone-inducible systems.
• To create a versatile genetic tool for precise control of gene expression in eukaryotic cells.
• To evaluate the system's efficiency and stringency in expressing foreign genes and reporter proteins.

Main Methods:

• Cloning Tet-On and mifepristone-inducible system elements into piggyBac-based trans- and cis-vectors.
• Inserting coding regions of RUNX1 splicing variants (RUNX1a, RUNX1b) into the cis-vector.
• Establishing transgenic 293T cells and inducing gene expression with doxycycline (DOX) and/or mifepristone.
• Analyzing gene and protein expression using qRT-PCR and Western blot.

Main Results:

• The piggyBac-based double-inducible system efficiently and stringently induced expression of reporter genes (GFP/mCherry) and co-expressed genes.
• Minimal mutual interference was observed between the induced genes.
• Successful expression of RUNX1 splicing variants was confirmed.
• The system demonstrated robust performance in transgenic 293T cells.

Conclusions:

• A novel piggyBac-based double-inducible system has been successfully developed.
• This system offers precise and stringent control over gene expression, suitable for studying gene function and interactions.
• The developed system is a valuable new genetic tool for in vitro and in vivo research across various organisms.