Abstract

The introduction and subsequent expression of foreign DNA inside living mammalian cells (transfection) is achieved by photoporation with a violet diode laser. We direct a compact 405 nm laser diode source into an inverted optical microscope configuration and expose cells to 0.3 mW for 40 ms. The localized optical power density of ~1200 MW/m2 is six orders of magnitude lower than that used in femtosecond photoporation (~104 TW/m2). The beam perforates the cell plasma membrane to allow uptake of plasmid DNA containing an antibiotic resistant gene as well as the green fluorescent protein (GFP) gene. Successfully transfected cells then expand into clonal groups which are used to create stable cell lines. The use of the violet diode laser offers a new and simple poration technique compatible with standard microscopes and is the simplest method of laser-assisted cell poration reported to date.

© 2005 Optical Society of America

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References

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Anal. Chem. (1)

J.S. Soughayer, T. Krasieva, S.C. Jacobson, J.M. Ramsey, B.J. Tromberg, and N.L. Allbritton, �??Characterization of cellular optoporation with distance,�?? Anal. Chem. 72, 1342-1347 (2000).
[CrossRef] [PubMed]

Biochem. and Biophys. Res. Commun. (1)

T.K. Wong and E. Neumann, �??Electric-field mediated gene-transfer,�?? Biochem. and Biophys. Res. Commun. 107, 584-587 (1982).
[CrossRef]

Biophys. J. (1)

Y Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman and B J. Tromberg, �??Evidence for localized cell heating induced by infrared optical tweezers,�?? Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Biotechnol. Lett. (1)

S.K. Mohanty, M. Sharma, and P.K. Gupta, �??Laser-assisted microinjection into targeted animal cells,�?? Biotechnol. Lett. 25, 895-899 (2003).
[CrossRef] [PubMed]

J. Biol. Chem. (1)

R. Fraley, S. Subramani, P. Berg, and D. Papahadjopolous, �??Introduction of liposome-encapsulated SV40 DNA into cells,�?? J. Biol. Chem. 255, 10431-10435 (1980).
[PubMed]

J. Biomed. Opt. (1)

H. Schneckenburger, A. Hendinger, R. Sailer, W.S.L. Strauss, and M. Schmitt, �??Laser-assisted optoporation of single cells,�?? J. Biomed. Opt. 7, 410-416 (2002).
[CrossRef] [PubMed]

J. Invest. Med. (1)

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, �??New technique for gene transfection using laser irradiation,�?? J. Invest. Med. 49, 184-190 (2001).
[CrossRef]

J. Mol. Appl. Genet. (1)

P. Southern and P. Berg, �??Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter,�?? J. Mol. Appl. Genet. 1, 327-341 (1982).
[PubMed]

J. Photochem. Photobiol. B-Biol. (1)

G. Palumbo, M. Caruso, E. Crescenzi, M.F. Tecce, G. Roberti, and A. Colasanti, �??Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation,�?? J. Photochem. Photobiol. B-Biol. 36, 41-46 (1996).
[CrossRef]

Mol. Therapy (1)

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, �??Femtosecond Infrared Laser - An Efficient and Safe in Vivo Gene Delivery System for Prolonged Expression,�?? Mol. Therapy 8, 342-350 (2003).
[CrossRef]

Nature (1)

U.K. Tirlapur and K. Konig, �??Targeted transfection by femtosecond laser,�?? Nature 418, 290-291 (2002).
[CrossRef] [PubMed]

Nature Genet. (1)

D.A. Rubinson, C.P. Dillon, A.V. Kwiatkowski, C. Sievers, L. Yang, J. Kopinja, M. Zhang, M.T. McManus, F.B. Gertler, M.L. Scott, and L. Van Parijs, �??A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference,�?? Nature Genet. 33, 401-406 (2003).
[CrossRef] [PubMed]

Opt. Express (2)

Plant J. (1)

U.K. Tirlapur and K. Konig, �??Femtosecond near-infrared laser pulses as a versatile non- invasive tool for intra-tissue nanoprocessing in plants without compromising viability,�?? Plant J. 31, 365-374 (2002).
[CrossRef] [PubMed]

Virology (1)

F.L. Graham and A.J. Van der Eb, �??A new technique for the assay of infectivity of human adenovirus 5 DNA,�?? Virology 52, 456-467 (1973).
[CrossRef] [PubMed]

Other (1)

D. McGloin and K. Dholakia, private communication

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Figures (4)

Fig. 1.
Fig. 1.

Photoporation apparatus. A violet diode laser (Toptica Photonics, 405 nm, 40 mW output power) is directed towards the sample through an inverted optical microscope setup.

Fig. 2.
Fig. 2.

Captured image of a cell being exposed to the focused violet diode laser beam (0.3 mW average power for 40 milliseconds).

Fig. 3.
Fig. 3.

Fluorescence images of cells transfected with an antibiotic-resistance gene and the GFP gene, taken several weeks after photoporation (images taken using FITC filter on a Zeiss Axioplan 2 imaging microscope). Cells are those that were subcultured from the antibiotic resistant colonies. GFP expression throughout the cells is clear. (a) Live cells (×20); (b) Fixed cells in Vectashield (×100).

Fig. 4.
Fig. 4.

Fluorescence images of live cells transfected with a plasmid containing an antibiotic-resistance gene and a gene encoding a red fluorescent protein (pDsRed-Mito, BD Biosciences, Oxford, U.K.), taken several weeks after photoporation. Expression of the red fluorescent protein in the cell mitochondria is clear. (a) ×20 magnification; (b) ×100 magnification

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