Optoinjection methods

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...

Reexamination Certificate

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C435S173100, C435S244000, C435S244000, C435S244000, C435S244000, C435S254800, C435S255300, C435S256300, C435S288700

Reexamination Certificate

active

06753161

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to methods for cell manipulation and more specifically to methods for transiently permeabilizing a cell so that a variety of exogenous materials, such as expressible foreign DNA, can be loaded into the cell.
Previous loading methods have included chemical treatments, microinjection, electroporation and particle bombardment. However, these techniques can be time-consuming and suffer from low yields or poor cell survival. Another technique termed “optoporation” has used light directed toward cells and the surrounding media to induce shock waves, thereby causing small holes to form temporarily in the surface of nearby cells, allowing materials to non-specifically enter cells in the area. Another technique termed “optoinjection” also uses light, but directs the light to specific cells. Nevertheless, previous light-based implementations techniques have suffered from the same disadvantages as other loading techniques.
Thus, there is a need for a method for rapid and efficient loading of a variety of exogenous molecules into cells, with high cell survival rates. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides optoinjection methods for transiently permeabilizing a target cell. In the general method, the steps are (a) illuminating a population of cells contained in a frame; (b) detecting at least one property of light directed from the frame; (c) locating a target cell by the property of light; and (d) irradiating the target cell with a pulse of radiation.
In particular embodiments, a static representation is obtained when the population of cells is substantially stationary; the cells are illuminated through a lens having a numerical aperture of at most 0.5; the pulse of radiation has a diameter of at least 10 microns at the point of contact with the target cell; or the resulting pulse of radiation delivers at most 1 &mgr;J/&mgr;m
2
. As a result, the method provides rapid and efficient loading of a variety of exogenous molecules into cells, with high cell survival rates.


REFERENCES:
patent: 5013660 (1991-05-01), Kasuya et al.
patent: 6143535 (2000-11-01), Palsson
Guo et al., “Laser-mediated Gene Transfer in Rice,”Physiol. Plantar., 93:19-24 (1995).
Krasieva et al., “Mechanisms of Cell Permeabilization by Laser Microirradiation,” LAMMP, Beckman Laser Institute, University of California at Irvine, Irvine, California, 8 pp., SPIE Proceedings (1999).
Palumbo et al., “Targeted Gene Transfer in Eucaryotic Cells by Dye-assisted Laser Optoporation,”J. Photochem. Photobiol., 36:41-46 (1996).
Soughayer et al., “Characterization of Cellular Optoporation with Distance,”Anal. Chem., 72:1342-1347 (2000).

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