Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-06-12
2001-04-24
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S283100, C435S285100, C435S285300, C435S286600
Reexamination Certificate
active
06221666
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to methods and apparatus for cytoplasmic loading using an impact-mediated procedure or “cell wounding” as a means of gaining direct entry to the cell protoplasm.
2. Description of the Related Art
A common approach in modern cell or molecular biology is to insert macro-molecules (e.g., dyes, proteins, nucleic acids) directly into the cytoplasm of living cells to monitor or modify specific cellular processes. Several existing techniques, such as electroporation and biolistics are used nearly exclusively for transfection (e.g., the loading of plasmid DNA constructs). Other transfection techniques, such as liposome fusion-based methods, exhibit limited transfection efficiencies, appear to work well with only a few cell types and have little utility for loading other macromolecules such as immunoglobulins or dyes. Microinjection, although the most direct and quantitative method of delivering a wide range of macromolecules to the cytoplasm of different cell types, is time consuming, requires specialized equipment and is impractical for loading large numbers of cells. Several simple and inexpensive cell-loading techniques, based on the use of mechanical force to transiently permeabilize the plasma membrane, achieve loading of adherent eukaryotic cells without the need for expensive and/or complex apparatus. These cell-loading techniques, scrape loading, scratch loading, bead loading, and syringe loading, have demonstrated the utility of “cell wounding” as a means of gaining entry to the cell cytoplasm.
A more detailed description of bead loading as a “cell wounding” technique is set forth in an article “Cytoplasmic Loading of Dyes, Protein and Plasmid DNA Using an Impact-Mediated Procedure” by Mark S. F. Clarke et al., BioTechniques, Vol.17, No.6 (1994), pp 1118-1125, which is incorporated herein by reference. A detailed description of the method of fluorescent flow cytometry to determine a mean fluorescence value is set forth in the article.
SUMMARY OF THE INVENTION
The present invention is directed to the method and apparatus for the cytoplasmic loading of macromolecules into living cells by an impact-mediated procedure that impacts the cells with a predetermined number of solid particles in a blast of propellant gas. More specifically, the present invention is directed to an impact-mediated procedure that is altered by gravitational conditions and is preferably carried out under hypergravity conditions.
Further, the present invention is directed to an IML method and apparatus that directs a blast of propellant gas through a rupturable membrane on which solid particles are supported, in order to achieve insertion of a predetermined number of particles into the propellant blast. Another preferred embodiment is a particle containment shell that includes a particle support membrane that ruptures at a predetermined propellant blast pressure and has a predetermined number of particles resting on the surface of the support membrane.
REFERENCES:
Clarke et al. Biotechniques. vol. 17(6), pp. 1118-1125, 1994.*
Takeuchi et al. Plant Molecular Biology. vol. 18, pp. 835-839, 1992.
Clarke Mark S. F.
Feeback Daniel L.
Lucas-Dean Rob G.
Vanderburg Charles R.
Withey Michael M.
Ketter James
Myers Kurt S.
Oceaneering International Inc.
Yucel Irem
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