High-throughput nuclear delivery and rapid expression of DNA via mechanical and electrical cell-membrane disruption
Nuclear transfection of DNA into mammalian cells is challenging yet critical for many biological and medical studies. Here, by combining cell squeezing and electric-field-driven transport in a device that integrates microfluidic channels with constrictions and microelectrodes, we demonstrate nuclear...
| Main Authors: | , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
2017
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| Online Access: | http://hdl.handle.net/1721.1/111022 https://orcid.org/0000-0003-4252-9335 https://orcid.org/0000-0003-4112-6622 https://orcid.org/0000-0002-9016-5962 https://orcid.org/0000-0003-4255-0492 https://orcid.org/0000-0001-7192-580X |
| Summary: | Nuclear transfection of DNA into mammalian cells is challenging yet critical for many biological and medical studies. Here, by combining cell squeezing and electric-field-driven transport in a device that integrates microfluidic channels with constrictions and microelectrodes, we demonstrate nuclear delivery of plasmid DNA within 1 h after treatment—the most rapid DNA expression in a high-throughput setting (up to millions of cells per minute per device). Passing cells at high speed through microfluidic constrictions smaller than the cell diameter mechanically disrupts the cell membrane, allowing a subsequent electric field to further disrupt the nuclear envelope and drive DNA molecules into the cytoplasm and nucleus. By tracking the localization of the endosomal sorting complex required for transport III protein CHMP4B (charged multivesicular body protein 4B), we show that the integrity of the nuclear envelope is recovered within 15 minutes of treatment. We also provide insight into subcellular delivery by comparing the performance of the disruption-and-field-enhanced method with those of conventional chemical, electroporation and manual-injection systems. |
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