| Autor: |
Matthew H. Wilson, Melissa A. Daniels, Kris M. Kahlig, Alfred L. George, Sai K. Saridey, Aparna Kaja |
| Rok vydání: |
2010 |
| Předmět: |
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| Zdroj: |
Biophysical Journal. 98:309a |
| ISSN: |
0006-3495 |
| DOI: |
10.1016/j.bpj.2009.12.1681 |
| Popis: |
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 co-express multiple transgenes or multi-protein complexes. 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 ∼60% (three transposons) or ∼30% (four transposons) co-expression of all delivered transgenes despite selection of a single marker transposon. We validated multiplexed piggyBac transposon delivery by co-expressing large transgenes encoding a multi-subunit 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. Co-transfection 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 concluded 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 multi-protein complexes. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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