Generation of cultured human cells stably expressing one or more recombinant 
gene sequences is a widely used approach in biomedical research, biotechnology, 
and drug development. Conventional methods are not efficient and have severe 
limitations especially when engineering cells to coexpress multiple transgenes 
or multiprotein complexes. In this report, we harnessed the highly efficient, 
nonviral, and plasmid-based piggyBac transposon system to enable concurrent 
genomic integration of multiple independent transposons harboring distinct 
protein-coding DNA sequences. Flow cytometry of cell clones derived from a 
single multiplexed transfection demonstrated approximately 60% (three 
transposons) or approximately 30% (four transposons) stable coexpression of all 
delivered transgenes with selection for a single marker transposon. We validated 
multiplexed piggyBac transposon delivery by coexpressing large transgenes 
encoding a multisubunit neuronal voltage-gated sodium channel (SCN1A) containing 
a pore-forming subunit and two accessory subunits while using two additional 
genes for selection. Previously unobtainable robust sodium current was 
demonstrated through 38 passages, suitable for use on an automated 
high-throughput electrophysiology platform. Cotransfection of three large (up to 
10.8 kb) piggyBac transposons generated a heterozygous SCN1A stable cell line 
expressing two separate alleles of the pore-forming subunit and two accessory 
subunits (total of four sodium channel subunits) with robust functional 
expression. We conclude that the piggyBac transposon system can be used to 
perform multiplexed stable gene transfer in cultured human cells, and this 
technology may be valuable for applications requiring concurrent expression of 
multiprotein complexes.