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.

What is claimed:  
     
         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 nucleotide 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 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 molecules 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.