High-throughput cell transfection device and methods of using thereof

Abstract

Transfecting biology cells with nucleic acid molecules (DNA, siRNA) is an essential prerequisite in elucidating how genes function in complex cellular context and how their activities could be modulated for therapeutic intervention. Traditionally studies are carried out on a low throughput gene-by-gene scale, which has created a huge bottleneck in functional genomic study and drug discovery. Development of high-throughput cell transfection technology will permit functional analysis of massive number of genes and how their activities could be modulated by chemical or biological entities inside cells. This invention describes design, construction of device and apparatus for high throughput effective cell transfection. Procedures and protocols for using the device and apparatus are also described in the application. Novel methods of using the device in cell-based assays are also disclosed.

1 . A method of identifying a transporter protein that affects transport of a compound in and/or out of a cell, comprising:
 a. providing multiple populations of cells, wherein upon transient transfection with a distinct foreign substance, each population of cells have altered expression of a distinct transporter protein as compared to other cell populations;   b. assaying the multiple populations of cells as early as three hours after said transient transfection for the ability of said distinct transporter expressed in said each population of cells to transport the compound in and/or out of a cell.   
     
     
         2 . The method of  claim 1 , wherein the step of providing comprises (i) culturing each population of cells in a distinct chamber supported by a porous membrane, and (ii) transiently transfecting said each population of cells with a vector encoding the distinct transporter protein by way of electroporation. 
     
     
         3 . The method of  claim 1 , wherein the step of providing comprises (i) culturing each population of cells in a distinct chamber supported by a porous membrane, and (ii) transiently transfecting said each population of cells with a siRNA targeting the distinct transporter protein by way of electroporation. 
     
     
         4 . The method of  claim 1 , wherein the transporter protein is selected from the group consisting of PEPT1, ASBT, OATP-B, P-gp, MRP2, and BCRP. 
     
     
         5 . The method of  claim 1 , wherein the transporter protein is an efflux transporter. 
     
     
         6 . The method of  claim 1 , wherein the transporter protein is an influx transporter. 
     
     
         7 . The method of  claim 1 , wherein the ability of said distinct transporter expressed in said each population of cells to transport the compound in and/or out of a cell is evidenced by a fluorescent or luminescent readout. 
     
     
         8 . The method of  claim 1 , wherein the multiple populations of cells are of the same type. 
     
     
         9 . The method of  claim 1 , wherein the multiple populations of cells are of different types. 
     
     
         10 . The method of  claim 1 , wherein the cells are adherent. 
     
     
         11 . The method of  claim 1 , wherein the cells are monolayer cells. 
     
     
         12 . The method of  claim 1 , wherein the cells are polarized. 
     
     
         13 . The method of  claim 1 , wherein the cells are selected from the group consisting of prostate cells, mammary cells, kidney cells, blood-brain-barrier cells, and liver cells. 
     
     
         14 . The method of  claim 1 , wherein the cells are cancer cells. 
     
     
         15 . The method of  claim 1 , where the cells are selected from the group consisting of MDCK (Madin-Darby Canine Kidney) cells, HEK293, and HepG2. 
     
     
         16 . The method of  claim 1 , wherein the cells are primary cells directly derived from tissues. 
     
     
         17 . The method of  claim 1 , wherein the cells are epithelial cells. 
     
     
         18 . The method of  claim 1 , wherein the assaying of multiple populations of cells is carried out within 6, 12, 24, 48 or 72 hours. 
     
     
         19 .- 33 . (canceled) 
     
     
         34 . A method of predicting a pharmacological property of a test compound, comprising:
 a. transiently transfecting multiple populations of cells, each population of which, with a distinct foreign substance to affect expression of a distinct transporter protein;   b. identifying from said transiently transfected populations of cells the transporter proteins that affect transport of test compound to generate a test compound/transporter interaction profile;   c. comparing said test compound/transporter interaction profile with that of a reference compound whose compound/transporter interaction profile and corresponding pharmacological property are characterized, thereby predicting the pharmacological property based on said comparison.   
     
     
         35 . The method of  claim 34 , further comprising modifying the structure of the test compound such that the test compound yields an altered test compound/transporter interaction profile as compared to that of the test compound without the modification. 
     
     
         36 . The method of  claim 34 , wherein the pharmacological property comprises ADME and pharmacokinetic properties. 
     
     
         37 . The method of  claim 34 , wherein the cells are adherent. 
     
     
         38 . The method of  claim 34 , wherein the cells are adherent monolayer cells. 
     
     
         39 . The method of  claim 34 , wherein the cells are polarized. 
     
     
         40 . The method of  claim 34 , wherein the transporter protein is selected from the group consisting of PEPT1, ASBT, OATP-B, P-gp, MRP2, and BCRP. 
     
     
         41 . The method of  claim 34 , wherein the reference compound is an approved or failed drug.