Chromosome-based platforms

Abstract

Artificial chromosomes, including ACes, that have been engineered to contain available sites for site-specific, recombination-directed integration of DNA of interest are provided. These artificial chromosomes provide tractable, efficient and rational engineering of the chromosome for a variety of applications.

1 . A platform artificial chromosome expression system (ACes) comprising one or a plurality of sites that participate in recombinase catalyzed recombination.  
     
     
         2 . The ACes of  claim 1  that contains a plurality of sites.  
     
     
         3 . The ACes of  claim 1  that is predominantly heterochromatin.  
     
     
         4 . The ACes of  claim 1  that is an animal ACes.  
     
     
         5 . The ACes of  claim 4  that is a mammalian Aces.  
     
     
         6 . The artificial chromosome expression system (ACes) of  claim 1 , wherein the recombinase and site(s) are from among the Cre/lox system of bacteriophage P1, the int/att system of lambda phage, the FLP/FRT system of yeast, the Gin/gix recombinase system of phage Mu, the Cin recombinase system, the Pin recombinase system of  E. coli  and the R/RS system of the pSR1 plasmid and any combination thereof.  
     
     
         7 . An isolated cell comprising, the chromosome of  claim 1 .  
     
     
         8 . The cell of  claim 7  that is an animal cell.  
     
     
         9 . The cell of  claim 7  that is a stem cell.  
     
     
         10 . The cell of  claim 7  that is a nuclear donor cell.  
     
     
         11 . The stem cell of  claim 9  that is human and is selected from among a mesenchymal stem cell, a hematopoietic stem cell, an adult stem cell and an embryonic stem cell.  
     
     
         12 . An animal artificial chromosomes comprising one or a plurality of att site(s), wherein: 
 an att site is heterologous to the chromosome; and    an att site permits site-directed integration in the presence of lambda integrase.    
     
     
         13 . The artificial chromosome of  claim 12  that is an artificial chromosome expression system (ACes).  
     
     
         14 . The chromosome of  claim 12 , wherein the att sites are selected from the group consisting of attP and attB or attL and attR, or variants thereof.  
     
     
         15 . The chromosome of  claim 13  that is an artificial chromosome that is predominantly heterochromatin.  
     
     
         16 . The chromosome of  claim 12  that is a mammalian artificial chromosome.  
     
     
         17 . The chromosome of  claim 13  that is a mammalian artificial chromosome.  
     
     
         18 . The chromosome of  claim 12 , wherein the integrase is a lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR  
     
     
         19 . A combination, comprising, the chromosome of  claim 1  and a first vector comprising the cognate recombination site, wherein the cognate recombination site is a site that recombines with the site engineered into the chromosome.  
     
     
         20 . The combination of  claim 19 , further comprising a nucleic acid molecule encoding a recombinase, wherein the nucleic acid molecule encoding the recombinase is provided on a second vector or on the first vector, or on the chromosome and its expression is under the control of an inducible promoter.  
     
     
         21 . The combination of  claim 20 , wherein a vector is the plasmid pCXLamIntR.  
     
     
         22 . The combination of  claim 20 , wherein a vector is the plasmid pDsRedN1-attB.  
     
     
         23 . A kit, comprising: 
 the combination of  claim 19;  and    optionally, instructions for introducing heterologous nucleic acid into the chromosome.    
     
     
         24 . An isolated cell comprising, the chromosome of  claim 12 .  
     
     
         25 . The cell of  claim 24 , wherein the cell is a stem cell.  
     
     
         26 . The cell of  claim 24 , wherein the cell is an animal cell.  
     
     
         27 . The chromosome of  claim 12 , wherein the integrase is a lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR.