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 permit tractable, efficient, rational engineering of the chromosome for a variety of applications.

1 . A plant chromosome 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.    
   
   
       2 . The chromosome of  claim 1  that is a plant artificial chromosome.  
   
   
       3 . The chromosome of  claim 1 , wherein the att sites are selected from the group consisting of attP and attB or attL and attR, or variants thereof.  
   
   
       4 . The chromosome of  claim 2  that is an artificial chromosome that is predominantly heterochromatin.  
   
   
       5 . 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.  
   
   
       6 . The combination of  claim 5 , further comprising nucleic acid encoding a recombinase, wherein the nucleic acid is on a second vector or on the first vector, or on the chromosome under an inducible promoter.  
   
   
       7 . The combination of  claim 6 , wherein the recombinase and sites are selected from 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 Pin recombinase system of  E. Coli,  the R/RS system of the pSR1 plasmid, or any combination thereof.  
   
   
       8 . The combination of  claim 6 , wherein a vector is the plasmid pCXLamIntR.  
   
   
       9 . The combination of  claim 5 , wherein a vector is the plasmid pDsRedN1-attB.  
   
   
       10 . A kit, comprising the combination of  claim 5  and optionally instructions for introducing heterologous nucleic acid into the chromosome.  
   
   
       11 . A cell comprising, the chromosome of  claim 1 .  
   
   
       12 . The cell of  claim 11 , wherein the cell is a stem cell.  
   
   
       13 . The cell of  claim 11 , wherein the cell is a plant cell.  
   
   
       14 . The chromosome of  claim 1 , wherein the integrase is a lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR.  
   
   
       15 . The chromosome of  claim 2  that is an artificial chromosome expression system (ACes).  
   
   
       16 . The chromosome of  claim 2  that is an artificial chromosome that contains no more than about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% euchromatin.  
   
   
       17 . A plant platform artificial chromosome expression system (ACes) comprising one or a plurality of sites that participate in a recombinase catalyzed recombination.  
   
   
       18 . A cell comprising the platform ACes of  claim 17 .  
   
   
       19 . The cell of  claim 18  that is an animal cell.  
   
   
       20 . The cell of  claim 19  that is a mammalian cell.  
   
   
       21 . The cell of  claim 19 , wherein the plant platform A Ces comprises transcriptional regulatory sequence of nucleotides derived from animals.  
   
   
       22 . The cell of  claim 20 , wherein the plant platform ACes comprises transcriptional regulatory sequence of nucleotides derived from animals.  
   
   
       23 . The cell of  claim 22 , wherein the regulatory sequence is selected from the group consisting of promoters, terminators, enhancers, silencers, and transcription factor binding sites.