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, 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 producing a cell that contains heterologous nucleic acid, comprising: 
 introducing a satellite artificial chromosome (SATAC) into the cell.    
     
     
         2 . The method of  claim 1 , wherein the cell is an animal cell.  
     
     
         3 . The method of  claim 2 , wherein the cell is a mammalian cell.  
     
     
         4 . The method of  claim 1 , wherein the SATAC is a mammalian artificial chromosome.  
     
     
         5 . The method of  claim 1 , wherein the SATAC is a plant artificial chromosome.  
     
     
         6 . The method of  claim 1 , wherein the SATAC is introduced by a method selected from direct DNA transfer or uptake, electroporation, lipofection, nuclear microinjection, liposome mediated transfer, microprojectile bombardment, polycation-mediated transfer and microcell fusion.  
     
     
         7 . The method of  claim 1 , wherein the SATAC is introduced by a method selected from microinjection, nuclear transfer, electrofusion, projectile bombardment and lipid-mediated transfer systems.  
     
     
         8 . The method of  claim 1 , wherein the SATAC is introduced into the cell by a method, comprising: 
 introducing a DNA fragment into the cell, wherein the DNA fragment comprises a selectable marker;    growing the cell under selective conditions to produce cells that have incorporated the DNA fragment into their genomic DNA; and    selecting a cell that comprises a satellite artificial chromosome (SATAC).    
     
     
         9 . The method of  claim 8 , wherein the DNA fragment is introduced into or adjacent to an amplifiable region of a chromosome in the cell.  
     
     
         10 . The method of  claim 9 , wherein the amplifiable region comprises rDNA.  
     
     
         11 . The method of  claim 9 , wherein the amplifiable region comprises heterochromatin.  
     
     
         12 . The method of  claim 8 , wherein the DNA is introduced into pericentric heterochromatin in a chromosome of the cell.  
     
     
         13 . The method of  claim 8 , wherein the method for producing the SATAC further comprises isolating the SATAC.  
     
     
         14 . The method of  claim 8 , wherein in the method for producing the SATAC, the DNA fragment comprises a sequence of nucleotides that targets the fragment to the heterochromatic region of a chromosome.  
     
     
         15 . The method of  claim 14 , wherein the targeting sequence of nucleotides comprises satellite DNA.  
     
     
         16 . The method of  claim 9 , wherein, in the method for producing the SATAC, the cell is a mammalian cell, a plant cell, a fish cell, an insect cell, a reptilian cell, an amphibian cell, an arachnid cell or a rodent cell.  
     
     
         17 . The method of  claim 1 , wherein the SATAC is an isolated substantially pure SATAC.  
     
     
         18 . The method of  claim 1 , wherein the SATAC is a megachromosome, comprising about 50 to about 450 megabases (Mb).  
     
     
         19 . The method of  claim 1 , wherein the SATAC is a megachromosome, comprising about 250 to about 400 Mb.  
     
     
         20 . The method of  claim 1 , wherein the SATAC is a megachromosome, comprising about 150 to about 200 Mb.  
     
     
         21 . The method of  claim 1 , wherein the SATAC is a megachromosome, comprising about 90 to about 120 Mb.  
     
     
         22 . The method of  claim 1 , wherein the SATAC is a megachromosome, comprising about 15 to about 60 Mb.  
     
     
         23 . The method of  claim 1 , wherein the cell is an embryonic cell.  
     
     
         24 . The method of  claim 1 , wherein the cell is an embryonic stem cell.  
     
     
         25 . The method of  claim 1 , wherein the SATAC comprises nucleic acid encoding a gene product or a plurality of gene products.  
     
     
         26 . The method of  claim 25 , wherein the gene products comprise a biosynthetic pathway.  
     
     
         27 . The method of  claim 2 , wherein the animal is a mammal, bird, fowl, fish, invertebrate, vertebrate, reptile or insect.  
     
     
         28 . The method of  claim 1 , wherein the cell is a mammalian cell, a plant cell, a fish cell, an insect cell, a reptilian cell, an amphibian cell, an arachnid cell or a rodent cell.