Multiplexed transposon-mediated stable gene transfer in human cells.
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.
Kahlig
Kristopher M
KM
Department of Medicine, Vanderbilt University, Nashville, TN 37235, USA.
Saridey
Sai K
SK
Kaja
Aparna
A
Daniels
Melissa A
MA
George
Alfred L
AL
Wilson
Matthew H
MH
D002460:Cell Line; D004251:DNA Transposable Elements; D015870:Gene Expression; D005819:Genetic Markers; D006801:Humans; D062550:NAV1.1 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames; D011485:Protein Binding; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
D004251:DNA Transposable Elements; D005819:Genetic Markers; D062550:NAV1.1 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; C568242:SCN1A protein, human; D015222:Sodium Channels