Transfection method and uses related thereto

Abstract

The invention features a method of identifying a nucleic acid molecule capable of post-transcriptional gene silencing by (a) affixing a plurality nucleic acid molecules onto a surface in discrete, defined locations; (b) contacting eukaryotic cells with the affixed nucleic acid molecules under appropriate conditions for entry of the nucleic acid molecules into the cells, whereby said nucleic acid molecules are introduced into the cells in the location in which they were affixed; and (c) determining the ability of the nucleic acid molecules to post-transcriptionally silence expression of a gene in the cells, wherein post-transcriptional gene silencing at a discrete, defined location identifies the nucleic acid molecules affixed at that location as being capable of post-transcriptional gene silencing.

What is claimed is:  
     
         1 . A method of identifying a nucleic acid molecule capable of post-transcriptional gene silencing, said method comprising the steps of: 
 (a) affixing a plurality nucleic acid molecules onto a surface in discrete, defined locations;    (b) contacting eukaryotic cells with the affixed nucleic acid molecules of step (a) under appropriate conditions for entry of the nucleic acid molecules into said eukaryotic cells; whereby said nucleic acid molecules are introduced into said eukaryotic cells in the location in which said nucleic acid molecules were affixed; and    (c) determining the ability of said nucleic acid molecules to post-transcriptionally silence expression of a gene in said cells, wherein post-transcriptional gene silencing at a discrete, defined location identifies the nucleic acid molecules affixed at said location as being capable of post-transcriptional gene silencing.    
     
     
         2 . The method of  claim 1 , wherein said nucleic acid molecules of step (a) are double-stranded RNA molecules or encode double-stranded RNA molecules.  
     
     
         3 . The method of  claim 1 , wherein each nucleic acid molecule to be introduced is contained in a vector.  
     
     
         4 . The method of  claim 3 , wherein the vector is an episomal vector or a chromosomally integrated vector.  
     
     
         5 . The method of  claim 3 , wherein the vector is a plasmid or a viral-based vector.  
     
     
         6 . The method of  claim 3 , wherein the vector is an expression vector.  
     
     
         7 . The method of  claim 1 , wherein said cells are mammalian cells.  
     
     
         8 . The method of  claim 1 , further comprising, between steps (a) and (b), the steps of: (i) covering the surface bearing the nucleic acid molecules with an appropriate amount of a transfection reagent and maintaining the resulting product under conditions appropriate for complex formation between the nucleic acid molecules and the transfection reagent; and (ii) removing the non-complexed transfection reagent.  
     
     
         9 . The method of  claim 1 , wherein the surface is glass, polystyrene, or plastic, optionally coated with poly-L-lysine.  
     
     
         10 . The method of  claim 1 , wherein the cells are plated at a density of 0.5×10 5 /cm 2  to 2.0×10 5 /cm 2 .  
     
     
         11 . The method of  claim 1 , wherein the cells are plated at a density of 0.5×10 5 /cm 2  to 1.0×10 5 /cm 2 .  
     
     
         12 . The method of  claim 1 , wherein said affixed plurality of nucleic acid molecules form an array of nucleic acid molecules and wherein said cells into which the nucleic acid molecules are introduced form an array of cells comprising the nucleic acid molecules.  
     
     
         13 . The method of  claim 12 , wherein the array comprises at least 96 different discrete, defined locations of known sequence composition.  
     
     
         14 . The method of  claim 13 , wherein the array comprises at least 192 different discrete, defined locations of known sequence composition.  
     
     
         15 . The method of  claim 14 , wherein the array comprises up to 10,000 to 15,000 different discrete, defined locations of known sequence composition.  
     
     
         16 . The method of  claim 1 , wherein each of the defined locations is 100-200 μm in diameter.  
     
     
         17 . The method of  claim 1 , wherein each of the defined locations is 200-500 μm apart from its nearest neighboring location.