Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1992-10-13
2003-01-07
Priebe, Scott D. (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S069100, C435S320100, C435S325000, C435S468000, C435S471000, C530S387100, C530S388850, C530S388200, C530S389200, C536S023100
Reexamination Certificate
active
06503755
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to method for transferring polynucleotide molecules into living cells by contacting the cells with small particles in the presence of a polynucleotide molecule-containing solution. The inventive method provides a rapid, simple procedure by which a large number of cells can be transfected with an efficiency comparable to that of existing transfection methods.
Gene transfer is an important technique in the genetic engineering of prokaryotic, eukaryotic and plant cells. Gene transfer has many applications, including for example, in human gene therapy, in the production of transgenic or genetically-improved livestock and plants, and in basic research.
Many physical methods have been described for the introduction of polynucleotide molecules into mammalian cells, including calcium phosphate co-precipitation, electric field-mediated gene transfer, direct microinjection, DEAE-dextran transfer, lipofection, fusion of erythrocytes or liposomes, osmotic lysis of pinosomes, centrifugation loading, and delivery of high velocity microprojectiles.
In the past decade, scientists have favored using a retrovirus to introduce exogenous genetic material into cells. In this method, a polynucleotide molecule or gene of interest to be transfected into a cell is first ligated within the genome of a retroviral vector. The retroviral vector containing the desired gene then uses the retrovirus's natural mechanism of infection to carry the gene into the cell. A disadvantage of this method is that the retroviral vector itself becomes an intracellular contaminant while carrying the target gene into the cell.
Three methods which directly introduce functional genetic material into living cells, and thereby avoid introducing extraneous viral genetic material into the cell, are electroporation, scrape-loading and bead loading. A common underlying mechanism between these methods is that each produces a transient disruption of the cell's plasma membrane, during which exogenous genetic molecules are able to diffuse inwardly.
Electroporation as described by Toneguzzo et al.,
Nucl. Acid Res.
16: 5515 (1988), provides a method for transfecting a large number of cells by delivering an electrical shock in order to temporarily disrupt membrane permeability. Although the electroporation method offers the advantage of direct DNA transfection into cells, there are several drawbacks to the method. Electroporation requires that a large number of cells be used, since 35-60% of the original number are killed by the electrical shock. Additionally, the electroporator machine is expensive, and the technique requires a minimum of about ½-2½ days to complete.
In the scrape-loading method of transfection, described by Fechheimer et al.,
Proc. Nat'l Acad. Sci. USA
84: 8463-67 (1987), a monolayer of cells adhered to a petri dish are contacted with a DNA loading solution. Next, the cells are loaded with DNA by gently scraping the cells with a blunted object. Scraped cells are removed from the petri dish and replated on another petri dish, grown in medium, and then assayed for gene expression.
In the scrape-loading method, replating the transfected cells takes one or two days, which is a serious disadvantage of the method. Consequently, the method cannot be used for investigations, for example, which utilize labels that degrade over such a lengthy time period. In addition, during scrape-loading, the cells undergo severe mechanical strain during the scraping action, which results in the loss of massive numbers of cells.
In achieving an analogous goal of loading molecules into living cells, the technique of “bead loading” described by McNeil et al.,
J. Cell Sci.
88: 669-77, (1987) was used. In this method, cells growing on a cover slip are exposed to a solution of macromolecules. Next, small glass beads are sprinkled over the surface of the cover slip, and gently rocked to evenly distribute the beads. The cover slips are washed, returned to culture medium, and examined for loading of the macromolecules.
Bead loading presents several advantages, including the ability to load large numbers of cells in only a few minutes time. Also, bead loading is performed on cells which are adhered to a surface, and does not require replating the cells after macromolecule loading. Bead loading of macromolecules causes less cell loss than does scrape-loading (McNeil et al.,
J. Cell Biol.
98: 1566-64 (1984). Thus, a method of transfecting polynucleotide molecules into cells which achieves many of the advantages offered by the bead loading technique for macromolecules is desired.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a method for transfecting adherent cells with polynucleotide molecules in just a few minutes time.
It is a further object to provide a method for serially transfecting a large amount of cells by recycling a polynucleotide/buffer solution used to particle transfect a first set of cells to particle transfect additional cells.
It is another object of the invention to provide an approach to transfection which achieves an efficiency of transfection and which maintains cell viability at levels comparable to other transfection methods.
It is a further object of the invention to provide a means of introducing polynucleotide molecules directly into cells which is performed easily and inexpensively without the need for expensive machinery or laborious techniques.
It is another object of the invention to particle transfect mammalian, bacterial, or plant cells (including haploid cells, protoplasts and haploid protoplasts).
In accomplishing these and other objects, there has been provided, in accordance with one aspect of the present invention, a method for transfecting polynucleotide molecules into cells, comprising the steps of providing a quantity of cells adhered to a support, contacting the cells with an amount of polynucleotide molecule, contacting the cells with particles and imparting kinetic energy to the particles such that contact between particles and cells occurs, whereby the cells are transfected with polynucleotide molecule to create transfected cells. A further step of detecting transfected cells which express the polynucleotide molecule also is provided.
Another object of the invention is to provide a method for transfecting polynucleotide molecules into cells which includes a recycling step. After performing particle transfection on a first set of cells, recycling comprises the steps of removing polynucleotide/buffer solution from the particle-transfected cells and bringing the solution into contact with a second set of cells adhered to a support, and transfecting the second set of cells.
Yet another object of the invention is to provide a method of adhering non-adherent cells to be transfected to a support, such as by contacting non-adherent cells with a ligand capable of binding to a support and to non-adherent cells to be transfected, and then performing particle transfection.
Another object of the invention is to provide a kit for use in transfecting a polynucleotide molecule into non-adherent cells, comprising a first receptacle containing a quantity of sterilized particles for use in particle transfection of cells and a second receptacle containing dehydrated buffer A.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides a rapid, inexpensive and efficient method for transfecting small or large-sized polynucleotide molecules into cells. A method within the present invention includes the steps of pr
Keating Armand
Matthews Kathryn E.
Mills Gordon B.
Foley & Lardner
Kaushal Sumesh
Keating Armand
Priebe Scott D.
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