Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes

Abstract

Methods for preparing cell lines that contain artificial chromosomes, methods for preparation of artificial chromosomes, methods for purification of artificial chromosomes, methods for targeted insertion of heterologous DNA into artificial chromosomes, methods for amplification of nucleic acids and methods for delivery of the chromosomes to selected cells and tissues are provided. Also provided are cell lines for use in the methods, and cell lines and chromosomes produced by the methods. Methods for use of the artificial chromosomes are also provided.

1 . A method for amplifying nucleic acid, comprising: 
 introducing a nucleic acid molecule into a plant cell, wherein the nucleic acid molecule includes a sequence of nucleotides that targets the nucleic acid molecule to an amplifiable region of a chromosome in the plant cell;    growing the plant cell; and    identifying from among the resulting plant cells those that include a chromosome with a portion that has undergone amplification.    
     
     
         2 . The method of  claim 1 , wherein the targeting sequence of nucleotides is selected from among those that target the molecule to the pericentric heterochromatic region of a chromosome.  
     
     
         3 . The method of  claim 1 , wherein the targeting sequence comprises rDNA.  
     
     
         4 . The method of  claim 1 , wherein the targeting sequence comprises an origin of replication or an amplification promoting sequence (APS).  
     
     
         5 . The method of  claim 1 , wherein the plant is tobacco, rice, maize, rye, soybean, wheat,  Brassica napus , cotton, lettuce, potato, tomato, petunia or  arabidopsis.    
     
     
         6 . The method of  claim 1 , wherein the amplified nucleic acid region includes amplified endogenous chromosomal nucleic acid.  
     
     
         7 . The method of  claim 1 , wherein the nucleic acid molecule encodes one or more genes.  
     
     
         8 . The method of  claim 1 , wherein the nucleic acid molecule encodes products that confer disease resistance to a plant.  
     
     
         9 . A method for amplifying a nucleic acid, comprising: 
 introducing a nucleic acid fragment comprising sequences of nucleotides targeted to an amplifiable region of a chromosome into a plant cell under conditions whereby the fragment integrates into the chromosome.    
     
     
         10 . The method of  claim 9 , further comprising replicating the plant cell.  
     
     
         11 . The method of  claim 9 , wherein the targeting sequences of nucleotides are selected from among those that target the molecule to the pericentric heterochromatic region of a chromosome.  
     
     
         12 . The method of  claim 9 , wherein the targeting sequences comprise rDNA.  
     
     
         13 . The method of  claim 9 , wherein the targeting sequences comprise an origin of replication or an amplification promoting sequence (APS).  
     
     
         14 . The method of  claim 9 , wherein the plant is tobacco, rice, maize, rye, soybean, wheat,  Brassica napus , cotton, lettuce, potato, tomato, petunia or  arabidopsis.    
     
     
         15 . A method for amplifying a nucleic acid, comprising: 
 introducing a nucleic acid fragment that comprises rDNA into a plant cell under conditions that produce plant cells that have incorporated the DNA fragment or a portion thereof that comprises the rDNA into a chromosome of the plant cell, whereby the nucleic acid fragment is amplified.    
     
     
         16 . The method of  claim 15 , further comprising replicating the plant cell.  
     
     
         17 . The method of  claim 15 , wherein the plant is tobacco, rice, maize, rye, soybean, wheat,  Brassica napus , cotton, lettuce, potato, tomato, petunia or  arabidopsis.    
     
     
         18 . A method for amplifying a nucleic acid, comprising: 
 introducing a nucleic acid fragment that comprises an origin of replication or an amplification promoting sequence into a plant cell under conditions to produce cells that have incorporated the DNA fragment or a portion thereof that comprises the origin of replication or an amplification promoting sequence into a chromosome of the cell, whereby incorporated DNA is amplified.    
     
     
         19 . The method of  claim 18 , wherein the plant is tobacco, rice, maize, rye, soybean, wheat,  Brassica napus , cotton, lettuce, potato, tomato, petunia or  Arabidopsis.    
     
     
         20 . The method of  claim 1 , wherein the plant is a protoplast.  
     
     
         21 . The method of  claim 1 , wherein the portin that has undergone amplification comprises the introduced nucleic acid molecule or a portion thereof.  
     
     
         22 . The method of  claim 1 , wherein the portion that has undergone amplification comprises centromeric nucleic acid.  
     
     
         23 . The method of  claim 1 , wherein the portion that has undergone amplification comprises pericentric heterochromatin.  
     
     
         24 . The method of  claim 1 , wherein the nucleic acid molecule that is introduced comprises heterologous nucleic acid.  
     
     
         25 . The method of  claim 1 , wherein the nucleic acid molecule that is introduced comprises a selectable marker.  
     
     
         26 . The method of  claim 24 , wherein the portion that has undergone amplification comprises the heterologous nucleic acid.  
     
     
         27 . A nucleic acid molecule, comprising: 
 nucleic acid encoding a gene product or gene products;    a selectable marker; and    sequences of nucleotides targeted to an amplifiable region of a chromosome in a cell.    
     
     
         28 . The nucleic acid molecule of  claim 27 , wherein the targeting sequences of nucleotides are selected from among those that target the molecule to the pericentric heterochromatic region of a chromosome.  
     
     
         29 . The nucleic acid molecule of  claim 27 , wherein the targeting sequences comprise rDNA.  
     
     
         30 . The nucleic acid molecule of  claim 27 , wherein the targeting sequences comprise an origin of replication or an amplification promoting sequence (APS).  
     
     
         31 . The nucleic acid molecule of  claim 27 , wherein the gene products encode a biosynthetic pathway.  
     
     
         32 . The nucleic acid molecule of  claim 27  that is a plasmid.  
     
     
         33 . The nucleic acid molecule of  claim 27 , wherein the cell is a plant cell.  
     
     
         34 . The nucleic acid molecule of  claim 33 , wherein the plant is tobacco, rice, maize, rye, soybean, wheat,  Brassica napus , cotton, lettuce, potato, tomato, petunia or  Arabidopsis.