Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes

Abstract

Methods for preparing cells 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 methods for producing transgenic plants and animals using the artificial chromosomes and the resulting transgenic organisms are provided.

What is claimed:  
     
         1 . A method for producing a transgenic non-human animal, comprising: 
 introducing a cell comprising a satellite artificial chromosome into a female non-human animal, wherein the cell develops into an embryo in a female non-human animal; and    allowing the embryo to develop into a transgenic non-human animal comprising a satellite artificial chromosome.    
     
     
         2 . The method of  claim 1 , wherein the satellite artificial chromosome comprises heterologous DNA that encodes a therapeutic product.  
     
     
         3 . The method of  claim 1 , wherein the cell contains the satellite artificial chromosome in a pronucleus.  
     
     
         4 . The method of  claim 1 , wherein the cell is a zygote.  
     
     
         5 . The method of  claim 1 , wherein the cell is a fertilized ovum.  
     
     
         6 . The method of  claim 1 , wherein the cell is an ovum that develops into an embryo or zygote.  
     
     
         7 . The method of  claim 1 , wherein the cell is a bird, mouse, reptile, amphibian, insect or fish cell.  
     
     
         8 . A method of producing a transgenic non-human animal embryo, comprising: 
 introducing a satellite artificial chromosome into a cell, wherein the cell develops in culture into a non-human animal embryo; and    culturing the cell under conditions whereby it develops into an embryo.    
     
     
         9 . The method of  claim 8 , wherein the cell comprises a fertilized oocyte, an ovum, a fertilized ovum or a zygote.  
     
     
         10 . The method of  claim 8 , wherein the cell is a bird, mouse, reptile, amphibian, insect, or fish cell.  
     
     
         11 . The method of  claim 1 , wherein the satellite artificial chromosome is isolated prior to introduction into the cell.  
     
     
         12  The method of  claim 1 , wherein the satellite artificial chromosome is introduced into the cell by a method selected from the group consisting of direct uptake, incubation with polyethylene glycol (PEG), lipofection, microinjection, cell fusion, microcell fusion, electroporation, electrofusion, particle bombardment, projectile bombardment, calcium phosphate precipitation and site-specific targeting.  
     
     
         13 . The method of  claim 1 , wherein the embryo and the female non-human animal are of the same species.  
     
     
         14 . A method for producing a transgenic non-human animal, comprising: 
 introducing an embryo comprising a satellite artificial chromosome into a female non-human animal; and    allowing the embryo to develop into a transgenic non-human animal comprising a satellite artificial chromosome.    
     
     
         15 . The method of  claim 14 , wherein: 
 the embryo is produced by introducing a satellite artificial chromosome into a cell that develops into an embryo; and    introduction is effected by a method selected from the group consisting of direct uptake, incubation with polyethylene glycol (PEG), lipofection, microinjection, cell fusion, microcell fusion, electroporation, electrofusion, particle bombardment, projectile bombardment, calcium phosphate precipitation and site-specific targeting.    
     
     
         16 . The method of  claim 15 , wherein the cell is selected from the group consisting of a bird, insect or fish cell.  
     
     
         17 . The method of  claim 15 , wherein the embryo and the female non-human animal are of the same species.  
     
     
         18 . A method for producing a transgenic non-human animal, comprising: 
 introducing a fertilized oocyte comprising a satellite artificial chromosome into a female non-human animal; and    allowing the resulting embryo to develop into a transgenic non-human animal comprising a satellite artificial chromosome.    
     
     
         19 . A method for producing a transgenic animal, comprising: 
 introducing an embryonic stem cell comprising a satellite artificial chromosome into an embryo;    introducing the embryo into a female mouse; and    allowing the embryo to develop into a transgenic animal comprising a satellite artificial chromosome.    
     
     
         20 . The method of  claim 19 , wherein the animal is a bird.  
     
     
         21 . A method for producing a transgenic non-human animal, comprising: 
 introducing an ovum comprising a satellite artificial chromosome (SATAC) into a female non-human animal, wherein the ovum develops into a zygote or embryo; and    allowing the embryo or zygote to develop into a transgenic non-human animal comprising the SATAC.    
     
     
         22 . The method of  claim 21 , wherein the satellite artificial chromosome is a megachromosome derived from a cell line having all of the identifying characteristics of the cell line deposited under ECACC accession number 96040928 or 96040929.