New technique for gene transfection using laser irradiation

Y. Shirahata, N. Ohkohchi, H. Itagak, S. Satomi

Research output: Contribution to journalArticlepeer-review

63 Citations (Scopus)

Abstract

Background: We have developed a gene transfection system using laser beams. The principle of this procedure is that a small hole is made in a cell membrane by pulse laser irradiation, and a gene contained in a medium is transferred into the cytoplasm through the hole. This hole disappears immediately with the application of laser irradiation of the appropriate power. Methods: A pulse-wave Nd:YAG laser with a wavelength of 355 nm was used to make a hole in a cell membrane. To trap a cell, a continuous-wave Nd:YAG laser with a wavelength of 1015 nm was used. Plasmids that encode the enhanced green fluorescent protein (EGFP) gene were contained in a medium and transferred to HuH-7 and NIH/3T3 cells with pulse laser irradiation. We evaluated transfection efficiency on the basis of the number of cells that expressed EGFP. Stimulatory protein 2 cells in suspension were fixed using a trapping laser and the neomycin-resistance gene was transfected by pulse laser irradiation. We examined cell proliferation in the selection medium. Results: Cells that expressed EGFP were recognized in the group that was irradiated by pulse laser. No cells expressed EGFP without irradiation. Transfection efficiency was ≈10% at a plasmid concentration of 10.0 μg/mL. At concentrations greater than 20 μg/mL, the transfection rate reached a plateau. We also successfully transfected neomycin-resistance genes to cells floating in suspension after fixation that was achieved with trapping laser irradiation. Conclusions: This method enables us to transfect targeted cells, ie, cells in suspension as well as attached cells, with a simple technique that does not involve harmful vectors. The present method is very useful for gene transfection in cellular biotechnology.

Original languageEnglish
Pages (from-to)184-190
Number of pages7
JournalJournal of Investigative Medicine
Volume49
Issue number2
DOIs
Publication statusPublished - 2001

Keywords

  • EGFP
  • Gene transfection
  • Laser beam
  • Targeted cells

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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