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 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.