Chromosome-based platforms
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.