| Surface transfection and expression procedure -> Monitor Keywords |
|
|
 
Surface transfection and expression procedureRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Process Of Mutation, Cell Fusion, Or Genetic Modification, Introduction Of A Polynucleotide Molecule Into Or Rearrangement Of Nucleic Acid Within An Animal CellSurface transfection and expression procedure description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060078996, Surface transfection and expression procedure. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application is a Continuation In Part Application, and claims priority to U.S. Provisional Applications Ser. No. 60/245,892 filed Nov. 3, 2000 and Ser. No. 60/305,552 filed Jul. 13, 2001, and to U.S. applications Ser. No. 09/960,454 filed Sep. 21, 2001, and Ser. No. 10/002,802 filed Nov. 2, 2001. FIELD OF THE INVENTION [0002] The present invention relates to a method of cell transfection, and in particular to the application of cells to nucleic acids which are immobilized on a surface and which then transfect the cells. In one embodiment, the nucleic acids are immobilized in an array. BACKGROUND OF THE INVENTION [0003] The wealth of information generated by the Human Genome Project and other genome projects has spurred research in many traditional disciplines such as cell biology and has given birth to entirely new disciplines such as bioinformatics and proteomics. The functional analysis of the nucleotide information provided by the Human Genome Project will fuel research questions over the next several decades and complete sequence determination of the human genome should be publicly available by 2003. This first step in characterization of the human genome presents tremendous opportunities to understand the function of these genes. [0004] An important extension of the various genome sequencing projects has been the sequencing of short sequences of nucleotides at the 5' and 3' ends of cDNA clones and the generation of expressed sequence tag (EST) sequences for comparison with the sequences obtained from genomic DNA (Gill and Sanseau (2000) Biotechnol Annu Rev 5:25-44). The presence of sequences within an EST database demonstrates that some portion of the gene is transcribed into mRNA in a particular cell and at some relative level of abundance. The sequencing of ESTs has provided substantial insight into the tissue specific and pathological regulation of gene expression. For many individual biomedical researchers, the partial characterization of ESTs has greatly facilitated the cloning and expression of genes of interest since many of the ESTs are readily available from public or commercial sources. [0005] A number of techniques currently under development to understand the regulation of gene expression take advantage of the large genomic databases and the availability of ESTs. One such major new technology is the use of DNA microarrays to study regulation of gene transcription by quantifying gene expression (Bittner et al. (1999) Nat Genet. 22(3):213-215; Graves D J (1999) Trends Biotechnol. 17(3):127-34; Watson and Akil (1999) Biol Psychiatry 45:533-543; Brown and Botstein (1999) Nat Genet 21:33-37; Duggan et al. (1999) Nat Genet 21:10-14; Young (2000) Cell 102:9-15). In this approach, very small amounts of DNA are applied to the surface of glass microscope slides (Schena et al. (1995) Science 270: 467-470). Typically, the DNA sample is a short PCR-amplified fragment corresponding to a known gene or EST sequence. Approximately 100 nanoliters of DNA solution containing 10 ng of DNA is applied and fixed to the glass slide. The application of DNA can be automated and robotic devices can spot 10,000 individual DNA samples onto a single microscope slide in arrays of easily identifiable patterns. Since the entire process is robotic, it is possible to make tens or hundreds of replicates of such slides. For the analysis of gene expression, the slides are hybridized with fluorescently labeled cDNA derived from mRNA preparations obtained from various samples. After washing, the amount of fluorescent DNA hybridized to the glass slide is indicative of the amount of mRNA complementary to the individual PCR fragment. The fluorescence intensity is quantitated using an array scanner to determine the fluorescence signal at the wavelengths of the fluorophores used to label the cDNA. [0006] This technique has been applied to the characterization of the transcriptional response of 8,600 individual genes in fibroblasts following serum stimulation (Iyer et al., 1999), and to the effect of viral infection, ionizing radiation, and cancer chemotherapeutic agents on transcriptional regulation (Brown and Botstein (1999) Nat Genet 21:33-37; Zhu H et al. (1998) Proc Natl Acad Sci U. S. A. 95(24):14470-5; Amundson S A et al. (1999) Oncogene 18(24):3666-72; Huang F et al. (1999) Oncogene 18(23):3546-52). [0007] Despite the wealth of information which potentially can be generated using arrayed DNA sequences, the information is limited to detecting the presence of nucleic acid sequences which are already present within a cell. Thus, DNA microarrays are currently used to determine gene expression. Once changes in transcription have been characterized, information about the relevant EST sequences is often limited to searching for homology to other known genes; even if such homology exists, the functionality of proteins encoded by the sequences is not known but can only be inferred. Thus, current methodologies are limited, as they do not provide any insight in the function of a particular gene, particularly those which encode proteins which do not show significant homology to known genes. Essential information for determining protein function, particularly of uncharacterized genes, requires expression of the protein and its characterization. An even greater limitation of the current techniques which employ microarrayed DNA is that major aspects of cellular regulation can not determined using such techniques, since most regulation of cell function occurs by modification of existing protein structure rather than by regulation of gene transcription. [0008] What is needed is the development of a high throughput screening assay for functional characterization of gene products; preferably, such a technique would also take advantage of the advances in DNA microarray technology. SUMMARY OF THE INVENTION [0009] Typically, determination of gene function involves transfection of cells with a gene under investigation. Currently, cell transfection is practiced by the addition of nucleic acid complexes to the media in which cells are grown; thus, there is no spatial restriction on the nucleic acid complexes which transfect the cells. It is an object of the present invention to provide a method that allows the functional characterization of proteins but that also takes advantage of the technological advances developed for DNA microarray hybridization. [0010] These objectives are met by the present invention, which provide a novel transfection method in which nucleic acids are spatially restricted before and at the initiation of transfection. Thus, the present invention provides a method in which cells are plated directly onto immobilized nucleic acids and transfected by the immobilized nucleic acids. The nucleic acids are immobilized on a surface on which the cells can be grown, and are restricted to the original area of immobilization under normal cell culture conditions. In some aspects of the present invention, the spatial arrangement of the nucleic acids is an array; in preferred embodiments, the array is a microarray. In some embodiments, the array is an ordered array; in other embodiments, the array is a random array. In preferred embodiments of the present invention, the microarrays are generated by DNA arrayers, which are readily commercially available. [0011] In one aspect, the method of the present invention further provides expression of the transfected nucleic acid; in yet an additional aspect, the method of the present further comprises detection of the expressed transfected nucleic acids. In this additional aspect of the present invention, the effects of transfected nucleic acids are easily measured, as for example by using appropriate fluorescent reporter constructs in the transfected cells, and detecting the fluorescence with commercially available scanners. The nucleic acids include, without being limited to, ESTs, PCR products, genomic DNA, cDNA, RNA, oligonucleotides and antisense constructs; such nucleic acids may be present within expression vectors. The present invention in its different aspects is referred to as Surface Transfection and Expression Procedure (STEP). [0012] Currently, STEP is immediately applicable to the numerous existing sets of ESTs, many of which are in eukaryotic expression vectors. Moreover, STEP can be utilized to take advantage of antisense techniques so that the function of a protein can be studied without the availability of a full-length cDNA. Like the differential hybridization to EST arrays, STEP is widely applicable to a variety of cellular regulation pathways and is an important and useful technique to bridge genomics and proteomics. [0013] Thus, the present invention provides a method of transfecting cells, comprising providing a transfection complex immobilized on a surface, the complex comprising nucleic acid and at least one complexing agent, and a cell; and contacting the cell with the nucleic acid in the immobilized transfection complex under conditions such that the cell is transfected. In some embodiments, the complexing agents are selected from the group consisting of ligands for receptors, DNA-binding molecules, and membrane permeable molecules. In other embodiments, the transfection complex comprises a first and second complexing agents, the first complexing agent comprising a ligand for receptors and the second complexing agent comprising a DNA binding molecule; in yet other embodiments, the transfection complex further comprises a third complexing agent, the third complexing agent comprising a membrane permeable molecule. In some preferred embodiments, the ligand is for a receptor which is endocytosed by cells, the DNA binding molecule is a cationic protein, and the membrane permeable molecule is a cationic lipid. In other preferred embodiments, the first complexing agent comprises transferrin and the second complexing agent comprises polylysine. In other preferred embodiments, the first complexing agent comprises viral protein, and the second complexing agent comprises polylysine or a histone; in even more preferred embodiments, the viral protein is selected from the group consisting of penton protein, HIV protein GP120, equine rhinitis A virus protein VP1, human adenovirus protein E3, and Epstein-Barr virus protein GP350, and the histone is histone 1A or histone 1B. In other embodiments, the transfection complex comprises at least two complexing agents, wherein at least two of the complexing agents are covalently linked to each other. In some preferred embodiments, the complexing agents comprise a ligand covalently linked to a cationic protein; in other preferred embodiments, the complexing agents comprise transferrin covalently linked to polylysine; in yet other preferred embodiments, the complexing agents comprise a viral protein covalently bound to polylysine or a histone. In yet other preferred embodiments, the transfection complex further comprises a third complexing agent, the third complexing agent comprising a membrane permeable molecule, which is preferably a cationic lipid. In yet other preferred embodiments, the complexing agents comprise transferrin, polylysine, and Lipofectamine.RTM., wherein transferrin is covalently linked to polylysine. In other embodiments, the transfection complex further comprises at least one additional agent selected from the group consisting of targeting molecules, transcription molecules, nucleic acid degradation inhibitors, and cell health and integrity modulators. In other embodiments, the nucleic acids are selected from the group consisting of ESTs, PCR products, genomic DNA, cDNA, RNA, oligonucleotides and antisense constructs; such nucleic acids may be present within expression vectors. In yet a further embodiment, at least one transfection complex comprises one type of nucleic acids. In another embodiment, at least one transfection complex comprises more than one type of nucleic acids. [0014] In another aspect of the present invention, the immobilized transfection complexes form an array of surface immobilized transfection complexes, wherein the transfection complexes comprise nucleic acids and at least one complexing agent. In some embodiments, the array is a microarray. In some embodiments, the array is ordered; in other embodiments, the array is random. In yet another aspect, the surface has a configuration including but not limited to flat, concave, convex, spherical, and cubical, and any and all combinations of these configurations. In some embodiments, the surface comprises at least two non-interconnected wells, such as indentations, troughs, chambers, depressions, reservoirs, or concavities; in particular embodiments, the surface comprises at least one well; and in preferred embodiments, the surface comprises wells as are found in a multi-well tissue culture plate; in individually preferred embodiments, the surface is a 96 well plate, a 384 well plate, or a 1536 well plate. In yet a further aspect, the surface includes but is not limited to a slide, a bead, a cube, a chip, a cube, a film, and a membrane. In another aspect of the present invention, the surface is made from a material which includes but is not limited to glass, plastic, films and membranes. In another aspect of the present invention, the surface is precoated with a compound to which both the nucleic acids and the cells adhere. In one embodiment, the compound includes but is not limited to polylysine, fibronectin, and lamenin. [0015] In other embodiments of the invention, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In other embodiments, the cells include but are not limited to cultured cells and cells freshly obtained from a source. In yet other embodiments, the cells are cultured cells that are selected from the group consisting of primary cultures, cell lines, and three-dimensional cultured cells. In yet further embodiments, the cells are in vivo; the cells include but are not limited to tissue cells, organ cells, and tumor cells. [0016] In another aspect of the present invention, the method further comprises the step of expressing the nucleic acids in the transfected cells. In a further aspect of the present invention, the method further comprises the step of detecting the expression of the nucleic acids in the transfected cells. In some embodiments, detecting the expression is monitored over a period of time. In other embodiments, detecting the expression is assayed in intact cells. In other embodiments, the nucleic acids encode at least one fluorescent reporter protein, and expression is detected by fluorescence microscopy. In yet other embodiments, the nucleic acids encode at least one luminescent reporter protein, and expression is detected by a light detector. [0017] The present invention also provides a method of transfecting a cell, comprising immobilizing a transfection complex on a surface, the complex comprising nucleic acid and at least one complexing agent, and contacting the cell with the immobilized nucleic acid in the transfection complex on the surface under conditions such that cells are transfected. The embodiments of the transfection complex, the form of the complexes immobilized on the surface, the surface, and the cells are as described above. In another aspect of the present invention, the method further comprises the step of expressing the nucleic acid in the transfected cells, and in a further aspect of the present invention, the method further comprises the step of detecting the expression of the nucleic acid in the transfected cells, with the embodiments as described above. [0018] The invention also provides a method of transfecting a cell, comprising combining nucleic acid with at least one complexing agent so as to form at least one transfection complex comprising the nucleic acid and the complexing agent; immobilizing the at least one transfection complex on a surface so as to form immobilized nucleic acid; and contacting a cell with the immobilized nucleic acid under conditions such that the cell is transfected. The embodiments of the transfection complex, the form of the transfection complexes immobilized on the surface, the surface, and the cells are as described above. In another aspect of the present invention, the method further comprises the step of expressing the nucleic acid in the transfected cell, and in a further aspect of the present invention, the method further comprises the step of detecting the expression of the nucleic acids in the transfected cell, with the embodiments as described above. [0019] The present invention also provides a method of transfecting a cell, comprising covalently linking transferrin to polylysine; combining nucleic acid and at least one cationic lipid with the covalently linked polylysine and transferrin so as to form a transfection complex; immobilizing the transfection complex on a surface so as to form immobilized nucleic acid; and contacting the cell with the immobilized nucleic acid so as to create a transfected cell. In further aspects of the invention, the method further comprises expressing the nucleic acid in the transfected cells; and in yet further aspects of the invention, the method further comprises the step of detecting the expression of the nucleic acids in the transfected cells. The embodiments of the transfection complexes, the form of the nucleic acids immobilized on the surface, the surface, and the cells are as described above. [0020] The present invention also provides a method of transfecting a cells, comprising providing transfection complexes immobilized on a surface in a random array, where the transfection complex comprises nucleic acid and at least one complexing agent, and a cell; and contacting the cell with the immobilized nucleic acids under conditions such that the cells is transfected. The embodiments of the transfection complexes, the form of the nucleic acids immobilized on the surface, the surface, and the cells are as described above. In another aspect of the present invention, the method further comprises the step of expressing the nucleic acid in the transfected cell, and in a further aspect of the present invention, the method further comprises the step of detecting the expression of the nucleic acid in the transfected cell, with the embodiments as described above. [0021] Another aspect of the present invention provides a method of immobilizing nucleic acid to a surface, comprising combining the nucleic acid with at least one complexing agent so as to form at least one transfection complex; and contacting the at least one transfection complex to the surface under conditions sufficient to immobilize the nucleic acid. The embodiments of the transfection complexes, the form of the nucleic acids immobilized on the surface, and the surface are as described above. The present invention also provides a surface comprising immobilized nucleic acids, wherein the nucleic acid is immobilized in at least one transfection complex, produced by any of the methods described above. Thus, in some embodiments, the surface comprises immobilized nucleic acids in an array of surface immobilized nucleic acids; in some preferred embodiments, the array is a microarray. In some embodiments, the array is ordered; in other embodiments, the array is random. The embodiments of the transfection complexes and the surface are as described above. Continue reading about Surface transfection and expression procedure... Full patent description for Surface transfection and expression procedure Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Surface transfection and expression procedure patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Surface transfection and expression procedure or other areas of interest. ### Previous Patent Application: Method of using fish plasma components for tissue engineering Next Patent Application: Methods for altering surface characteristics of microspheres Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Surface transfection and expression procedure patent info. IP-related news and info Results in 0.29423 seconds Other interesting Feshpatents.com categories: Software: Finance , AI , Databases , Development , Document , Navigation , Error 174 |
 * Protect your Inventions * US Patent Office filing 
PATENT INFO |
|
