Reverse transfection method

Details Reverse transfection method Reverse transfection method

2000-09-18

2003-04-08

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Process of mutation, cell fusion, or genetic modification

Introduction of a polynucleotide molecule into or...

C435S320100, C435S440000, C435S006120, C435S007210, C435S174000

Reexamination Certificate

active

06544790

ABSTRACT:

BACKGROUND OF THE INVENTION
Genome and expressed sequence tag (EST) projects are rapidly cataloging and cloning the genes of higher organisms, including humans. The emerging challenge is to uncover the functional roles of the genes and to quickly identify gene products with desired properties. The growing collection of gene sequences and cloned cDNAs demands the development of systematic and high-throughput approaches to characterizing the gene products. The uses of DNA microarrays for transcriptional profiling and of yeast two-hybrid arrays for determining protein-protein interactions are recent examples of genomic approaches to the characterization of gene products (Schena, M., et al.,
Nature
, 10:623 (2000)). Comparable strategies do not exist to analyze the function, within mammalian cells, of large sets of genes. Currently, in vivo gene analysis can be done—on a gene-by-gene scale—by transfecting cells with a DNA construct that directs the overexpression of the gene product or inhibits its expression or function. The effects on cellular physiology of altering the level of a gene product is then detected using a variety of functional assays.
A variety of DNA transfection methods, such as calcium phosphate coprecipitation, electroporation and cationic liposome-mediated transfection (e.g., lipofection) can be used to introduce DNA into cells and are useful in studying gene regulation and function. Additional methods, particularly high throughput assays that can be used to screen large sets of DNAs to identify those encoding products with properties of interest, would be useful to have available.
SUMMARY OF THE INVENTION
Described herein is a strategy for the high throughput analysis of gene function in mammalian cells. A method to create transfected cell microarrays that are suitable for rapidly screening large sets of cDNAs or DNA constructs for those encoding desired products or for causing cellular phenotypes of interest is described. Using a slide printed with sets of cDNAs in expression vectors, a living microarray of cell clusters expressing the gene products has been generated. The cell clusters can be screened for any property detectable on a surface and the identity of the responsible cDNA(s) determined form the coordinates of the cell cluster with a phenotype of interest.
Accordingly, the present invention relates to a method, referred to as a reverse transfection method, in which a defined nucleic acid (a nucleic acid of known sequence or source), also referred to as a nucleic acid of interest or a nucleic acid to be introduced into cells, is introduced into cells in defined areas of a lawn of eukaryotic cells, in which it will be expressed or will itself have an effect on or interact with a cellular component or function. Any suitable nucleic acid such as an oligonucleotide, DNA and RNA can be used in the methods of the present invention. The particular embodiments of the invention are described in terms of DNA. However, it is to be understood that any suitable nucleic acid is encompassed by the present invention.
In one embodiment, the present invention relates to a method in which defined DNA (DNA of known sequence or source), also referred to as DNA of interest or DNA to be introduced into cells, is introduced into cells in defined areas of a lawn of eukaryotic cells, in which it will be expressed or will itself have an effect on or interact with a cellular component or function. In the method, a mixture, defined below, comprising DNA of interest (such as cDNA or genomic DNA incorporated in an expression vector) and a carrier protein is deposited (e.g., spotted or placed in small defined areas) onto a surface (e.g., a slide or other flat surface, such as the bottoms of wells in a multi-welled plate) in defined, discrete (distinct) locations and allowed to dry, with the result that the DNA-containing mixture is affixed to the surface in defined discrete locations.
Such locations are referred to herein, for convenience, as defined locations. The DNA-containing mixture can be deposited in as many discrete locations as desired. The resulting product is a surface bearing the DNA-containing mixture in defined discrete locations; the identity of the DNA present in each of the discrete locations (spots) is known/defined. Eukaryotic cells, such as mammalian cells (e.g., human, monkey, canine, feline, bovine, or murine cells), bacterial, insect or plant cells, are plated (placed) onto the surface bearing the DNA-containing mixture in sufficient density and under appropriate conditions for introduction/entry of the DNA into the eukaryotic cells and expression of the DNA or its interaction with cellular components. Preferably, the eukaryotic cells (in an appropriate medium) are plated on top of the dried DNA-containing spots at high density (e.g., 1×10
5
/cm
2
), in order to increase the likelihood that reverse transfection will occur. The DNA present in the DNA-containing mixture affixed to the surface enters eukaryotic cells (reverse transfection occurs) and is expressed in the resulting reverse transfected eukaryotic cells.
In one embodiment of the method, referred to as a “gelatin-DNA” embodiment, the DNA-containing mixture, referred to herein as a gelatin-DNA mixture, comprises DNA (e.g., DNA in an expression vector) and gelatin, which is present in an appropriate solvent, such as water or double deionized water. The mixture is spotted onto a surface, such as a slide, thus producing a surface bearing (having affixed thereto) the gelatin-DNA mixture in defined locations. The resulting product is allowed to dry sufficiently that the spotted gelatin-DNA mixture is affixed to the slide and the spots remain in the locations to which they have become affixed, under the conditions used for subsequent steps in the method. For example, a mixture of DNA in an expression vector and gelatin is spotted onto a slide, such as a glass slide coated with &Sgr; poly-L-lysine (e.g., Sigma, Inc.), for example, by hand or using a microarrayer. The DNA spots can be affixed to the slide by, for example, subjecting the resulting product to drying at room temperature, at elevated temperatures or in a vacuum-dessicator. The length of time necessary for sufficient drying to occur depends on several factors, such as the quantity of mixture placed on the surface and the temperature and humidity conditions used.
The concentration of DNA present in the mixture will be determined empirically for each use, but will generally be in the range of from about 0.01 &mgr;g/&mgr;l to about 0.2 &mgr;g/&mgr;l and, in specific embodiments, is from about 0.02 &mgr;g/&mgr;l to about 0.10 &mgr;g/&mgr;l. Alternatively, the concentration of DNA present in the mixture can be from about 0.01 &mgr;g/&mgr;l to about 0.5 &mgr;g/&mgr;l, from about 0.01 &mgr;g/&mgr;l to about 0.4 &mgr;g/&mgr;l and from about 0.01 &mgr;g/&mgr;l to about 0.3 &mgr;g/&mgr;l . Similarly, the concentration of gelatin, or another carrier macromolecule, can be determined empirically for each use, but will generally be in the range of 0.01% to 0.5% and, in specific embodiments, is from about 0.05% to about 0.5%, from about 0.05% to about 0.2% or from about 0.1% to about 0.2%. The final concentration of DNA in the mixture (e.g., DNA in gelatin) will generally be from about 0.02 &mgr;g/&mgr;l to about 0.1 &mgr;g/&mgr;l and in a specific embodiment described herein, DNA is diluted in 0.2% gelatin (gelatin in water) to produce a final concentration of DNA equal to approximately 0.05 &mgr;g/&mgr;l.
If the DNA used is present in a vector, the vector can be of any type, such as a plasmid or viral-based vector, into which DNA of interest (DNA to be expressed in reverse transfected cells) can be introduced and expressed (after reverse transfection) in recipient cells. For example, a CMV-driven expression vector can be used. Commercially available plasmid-based vectors, such as pEGFP (Clontech) or pcDNA3 (Invitrogen), or viral-based vectors can be used. In this embodiment, after drying of the spots containing the gelatin-DNA mixture, the surface bear

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