Multiplexed transposon-mediated stable gene transfer in human cells.

Area of Science:

• Molecular Biology
• Cell Biology
• Biotechnology

Background:

• Stable expression of recombinant genes in human cells is crucial for research and drug development.
• Conventional gene delivery methods face challenges, particularly for coexpressing multiple genes or complex protein assemblies.
• The piggyBac transposon system offers a nonviral, plasmid-based approach for gene integration.

Purpose of the Study:

• To develop and validate a multiplexed piggyBac transposon system for concurrent genomic integration of multiple genes.
• To assess the efficiency of stable coexpression of several transgenes in cultured human cells.
• To demonstrate the system's capability in expressing large, functional multiprotein complexes, such as voltage-gated sodium channels.

Main Methods:

• Utilized a multiplexed piggyBac transposon system for simultaneous delivery of multiple independent transposons.
• Performed single multiplexed transfections in cultured human cells.
• Employed flow cytometry for assessing stable coexpression of transgenes.
• Validated the system by coexpressing a multisubunit neuronal voltage-gated sodium channel (SCN1A) and selection genes.

Main Results:

• Achieved approximately 60% stable coexpression for three transposons and 30% for four transposons with single-marker selection.
• Demonstrated robust and stable functional expression of a multisubunit sodium channel (SCN1A) for 38 passages.
• Successfully generated a stable cell line coexpressing four sodium channel subunits (two alleles of pore-forming and two accessory subunits) from three large transposons.

Conclusions:

• The piggyBac transposon system enables efficient multiplexed stable gene transfer in cultured human cells.
• This technology is valuable for applications requiring concurrent expression of multiprotein complexes.
• The developed system overcomes limitations of conventional methods for complex gene engineering.