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 method of introducing one or more heterologous nucleic acid(s) into an artificial chromosome, wherein the artificial chromosome is a mammalian artificial chromosome and is predominantly heterochromatin, the method comprising:
 (a) introducing one or a plurality of recombination site(s) comprising a heterologous att site into the artificial chromosome   (b) mixing the artificial chromosome containing the one or a plurality of recombination site(s) comprising a heterologous att site with a first vector comprising the heterologous nucleic acid and one or a plurality of cognate recombination site(s), wherein the cognate recombination site(s) is a site that participates in recombinase catalyzed recombination with att site(s) in the artificial chromosome;   (c) incubating the resulting mixture in the presence of at least one lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR under conditions whereby recombination between the att site and cognate recombination site is effected, thereby introducing the heterologous nucleic acid into the artificial chromosome.   
     
     
         2 . The method of  claim 1 , wherein the artificial chromosome contains a plurality of att sites. 
     
     
         3 .- 13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the att sites are selected from the group consisting of attP with attB and attL with attR. 
     
     
         15 .- 19 . (canceled) 
     
     
         20 . The method of  claim 1 , wherein the lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR is encoded by a nucleic acid molecule, wherein the nucleic acid molecule is provided on a second vector or on the first vector, or on the artificial chromosome and its expression is under the control of an inducible promoter. 
     
     
         21 . The method of  claim 20 , wherein the second vector is the plasmid pCXLamIntR. 
     
     
         22 . The method of  claim 20 , wherein the first vector is the plasmid pDsRedN1-attB. 
     
     
         23 .- 27 . (canceled) 
     
     
         28 . The method of  claim 1  wherein the one or a plurality of recombination site(s) is introduced into the artificial chromosome together with a selectable marker. 
     
     
         29 . The method of  claim 28  wherein the selectable marker is selected from the group consisting of a gene that provides a selective growth advantage, an antibiotic resistance gene, and a gene encoding a detectable protein, wherein the detectable protein is chromogenic, fluorescent, or capable of being bound by an antibody and FACs sorted. 
     
     
         30 . The method of  claim 29  wherein the selectable marker is a puromycin-resistance gene. 
     
     
         31 . The method of  claim 28  wherein the one or a plurality of recombination site(s) and a selectable marker are comprised in pSV40-193attPsensePur ( FIG. 4 , SEQ ID NO:113).