Method of transfecting cells by electroporation and apparatus for same

Abstract

An electroporation apparatus for introducing exogenous material into cells is described herein. The apparatus comprises first a base member configured for holding a cell support, the cell support having a top surface portion, with the top surface portion configured for carrying adherent cells. The apparatus further comprises an electrode carrier operably associated with the base member, the electrode carrier having a bottom surface portion, a first electrode connected to the electrode carrier, and a second electrode also connected to the electrode carrier. The electrode carrier has a channel formed therein, with the channel positioned between the first electrode and the second electrode, so that exogenous material may be introduced through the channel and into contact with the cells. Methods for introducing exogenous compounds into a cell and for visually detecting the location of binding events within a cell are also disclosed.

1 . A method for screening drugs by visually detecting the location of binding events within a cell comprising: 
 introducing a fusion protein into said cell, said fusion protein comprising a binding domain and a detectable domain;    detecting the location of increased levels of said detectable domain within said cell, the location of increased levels of the detectable domain indicating the location of a compound within said cell to which said binding domain specifically binds; and then    identifying the drugs which affect the location of the increased levels of said detectable domain within the cell.    
     
     
         2 . A method according to  claim 1 , wherein said introducing step is carried out by introducing RNA encoding said fusion protein into said cell, wherein said fusion protein is translated from said RNA.  
     
     
         3 . A method according to  claim 1 , wherein said detectable domain comprises a fluorescent protein.  
     
     
         4 . A method according to  claim 1 , wherein said introducing step is carried out by electroporation.  
     
     
         5 . A method according to  claim 1 , wherein said introducing step is carried out by microparticle bombardment.  
     
     
         6 . A method according to  claim 1 , wherein the method is an assay for tyrosine phosphorylation wherein said binding domain is tyrosine kinase Syk or a fragment thereof that binds to the F cε RI receptor.  
     
     
         7 . A method according to  claim 1 , wherein the method is an assay for diacylglycerol wherein said binding domain is the C 1  fragment of protein kinase C or a fragment of said C 1  fragment that binds to diacylglycerol.  
     
     
         8 . A method according to  claim 1 , wherein the method is an assay for nuclear translocation or transcriptional activation wherein said binding domain is CaM-Kinase IV.  
     
     
         9 . A method according to  claim 1 , wherein the method is an assay for cell secretion wherein said binding domain is catalase.  
     
     
         10 . A method according to  claim 3 , wherein the fluorescent protein is a Green Fluorescent Protein derived from Aequorea victoria, and analogues and derivatives thereof.  
     
     
         11 . A method for screening drugs by visually detecting the location of binding events within a cell comprising: 
 introducing a fusion protein into said cell, said fusion protein comprising a binding domain and a detectable domain, wherein said detectable domain is a Green Fluorescent Protein derived from  Aequorea victoria,  and analogues and derivatives thereof;    detecting the location of increased levels of said detectable domain within said cell, the location of increased levels of the detectable domain indicating the location of a compound within said cell to which said binding domain specifically binds; and then    identifying the drugs which affect the location of the increased levels of said detectable domain within the cell.    
     
     
         12 . A method according to  claim 11 , wherein said introducing step is carried out by introducing RNA encoding said fusion protein into said cell, wherein said fusion protein is translated from said RNA.  
     
     
         13 . A method according to  claim 11 , wherein said introducing step is carried out by electroporation.  
     
     
         14 . A method according to  claim 11 , wherein said introducing step is carried out by microparticle bombardment.  
     
     
         15 . A method according to  claim 11 , wherein the method is an assay for tyrosine phosphorylation wherein said binding domain is tyrosine kinase Syk or a fragment thereof that binds to the F cε RI receptor.  
     
     
         16 . A method according to  claim 11 , wherein the method is an assay for diacylglycerol wherein said binding domain is the C 1  fragment of protein kinase C or a fragment of said C 1  fragment that binds to diacylglycerol.  
     
     
         17 . A method according to  claim 11 , wherein the method is an assay for nuclear translocation or transcriptional activation wherein said binding domain is CaM-Kinase IV.  
     
     
         18 . A method according to  claim 11 , wherein the method is an assay for cell secretion wherein said binding domain is catalase.