Methods for delivering nucleic acid molecules into cells and assessment thereof

Abstract

Methods for delivering nucleic acid molecules into cells and methods for measuring nucleic acid delivery into cells and the expression of the nucleic acids are provided. The methods are designed for introduction of large nucleic acid molecules, including artificial chromosomes, into cells, and are practiced in vitro and in vivo.

1 . A method for detecting or determining delivery and expression of nucleic acid molecules introduced into a cell comprising; 
 introducing a labelled large nucleic acid molecules that encode a reporter gene into a cell;    detecting labelled cells as an indication of delivery of the nucleic acid into the cell; and    measuring the product of the reporter gene as an indication of DNA expression of the labelled large nucleic acid molecule in the cell, whereby delivery into and expression of the nucleic acid molecule in the cell is detected or determined.    
     
     
         2 . The method of  claim 1 , wherein the labelled cells are detected by flow cytometry, fluorimetry, cell imaging or fluorescence spectroscopy.  
     
     
         3 . The method of  claim 1 , wherein the labelled cells are detected by flow cytometry.  
     
     
         4 . The method of  claim 1 , wherein the nucleic acid molecule is DNA.  
     
     
         5 . The method of  claim 1 , wherein the label is selected from the group consisting of a nucleotide analog, a nucleoside analog and a ribonucleoside analog.  
     
     
         6 . The method of  claim 1 , wherein the label is a thymidine analog.  
     
     
         7 . The method of  claim 1 , wherein the label is iododeoxyuridine (IdU or IdUrd) or bromodeoxyuridine (BrdU).  
     
     
         8 . The method of  claim 1 , wherein the reporter gene encodes a fluorescent protein, or enzyme or antibody.  
     
     
         9 . The method of  claim 8 , wherein the enzyme is a luciferase, β-galactosidase or alkaline phosphatase.  
     
     
         10 . The method of  claim 8 , wherein the fluorescent protein is a red, green or blue fluorescent protein.  
     
     
         11 . The method of  claim 8 , step (a) comprises contacting the nucleic acid molecule is with a delivery agent that comprises a cationic compound.  
     
     
         12 . The method of  claim 11 , wherein the compound is selected from the group consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride (DOTMA), dioleoylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), dioleoyl phosphatidylethanolamine (DOPE), C 52 H 106 N 6 O 4 .4CF 3 CO 2 H, C 88 H 178 N 8 O 4 S 2 .4CF 3 CO 2 H, C 40 H 84 NO 3 P.CF 3 CO 2 H, C 50 H 103 N 7 O 3 .4CF 3 CO 2 H, C 55 H 116 N 8 O 2 .6CF 3 CO 2 H, C 49 H 102 N 6 O 3 .4CF 3 CO 2 H, C 44 H 89 N 5 O 3 .2CF 3 CO 2 H, C 41 H 78 NO 8 P), C 100 H 206 N 12 O 4 S 2 .8CF 3 CO 2 H, C 162 H 330 N 22 O 9 .13CF 3 CO 2 H, C 43 H 88 N 4 O 2 .2CF 3 CO 2 H, C 43 H 88 N 4 O 3 .2CF 3 CO 2 H and (1-methyl-4-(1-octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride.  
     
     
         13 . The method of  claim 12 , wherein the nucleic acid molecule is a natural chromosomes, an artificial chromosome, a fragment of a chromosome or a naked DNA that is greater than at least about 0.6 megabase in size.  
     
     
         14 . The method of  claim 1 , wherein the nucleic acid molecule is an artificial chromosome, a plasmid, a chromosome fragment, a naked DNA, or a natural chromosome.  
     
     
         15 . The method of  claim 1 , wherein the nucleic acid molecule is an artificial chromosome expression system (ACe).  
     
     
         16 . The method of  claim 1 , wherein the cell is a eukaryotic cell.  
     
     
         17 . The method of  claim 16 , wherein the cell is a primary cell, a cell line, a plant cell, or an animal cell.  
     
     
         18 . The method of  claim 16 , wherein the cell is a stem cell, a nuclear transfer donor cell, a tumor cell or a transformed cell.  
     
     
         19 . The method of  claim 1 , wherein the cell is selected from the group consisting of a primary cell, an immortalized cell, an embryonic cell, a stem cell, a transformed cells and a tumor cell.