Chromosome-based platforms
First Claim
Patent Images
1. A method of introducing heterologous nucleic acid into a chromosome, comprising:
- contacting a eukaryotic chromosome comprising one or a plurality of att site(s) with a nucleic acid molecule comprising both the heterologous nucleic acid and a recombination site, in the presence of a recombinase that promotes recombination between the sites in the chromosome and in the nucleic acid molecule, wherein;
the one or a plurality of att site(s) are heterologous to the chromosome; and
the one or a plurality of att site(s) permits site-directed integration in the presence of lambda integrase.
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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.
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Citations
85 Claims
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1. A method of introducing heterologous nucleic acid into a chromosome, comprising:
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contacting a eukaryotic chromosome comprising one or a plurality of att site(s) with a nucleic acid molecule comprising both the heterologous nucleic acid and a recombination site, in the presence of a recombinase that promotes recombination between the sites in the chromosome and in the nucleic acid molecule, wherein;
the one or a plurality of att site(s) are heterologous to the chromosome; and
the one or a plurality of att site(s) permits site-directed integration in the presence of lambda integrase. - View Dependent Claims (2, 3, 4, 45, 46)
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5. A method for introducing heterologous nucleic acid into a platform artificial chromosome, comprising:
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(a) mixing an artificial chromosome comprising at least a first recombination site and a vector comprising at least a second recombination site and the heterologous nucleic acid;
(b) incubating the resulting mixture in the presence of at least one recombination protein under conditions whereby recombination between the first and second recombination sites is effected, thereby introducing the heterologous nucleic acid into the artificial chromosome. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 29, 30, 31, 32, 47)
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- 22. A human mesenchymal cell comprising an artificial chromosome.
- 26. A lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR.
- 33. A method for producing a transgenic animal, comprising introducing a platform-ACes into an embryonic cell.
- 40. A cell line useful for making a library of ACes, comprising a multiplicity of heterologous recombination sites randomly integrated throughout the endogenous chromosomes.
- 42. A library of cells useful for genomic screening, said library comprising a multiplicity of cells, wherein each cell comprises an ACes having a mutually exclusive portion of a chromosomal nucleic acid therein.
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44. A method of making one or more cell lines, comprising
a) integrating into endogenous chromosomal DNA of a selected cell species, a multiplicity of heterologous recombination sites, b) introducing a multiplicity of ACes under conditions that promote the site-specific chromosomal arm exchange of the ACes into, and out of, a multiplicity of the heterologous recombination sites integrated within the cell'"'"'s endogenous chromosomal DNA; -
c) isolating said multiplicity of ACes, thereby producing a library of ACes whereby a multiplicity of ACes have mutually exclusive portions of the endogenous chromosomal DNA therein;
d) introducing the isolated multiplicity of ACes of step c) into a multiplicity of cells, thereby creating a library of cells;
e) selecting different cells having mutually exclusive ACes therein and clonally expanding or differentiating said different cells into clonal cell cultures, thereby creating one or more cell lines.
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- 50. A modified iron-induced promoter comprising SEQ ID NO:
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52. A vector, comprising:
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a recognition site for recombination; and
a sequence of nucleotides that targets the vector to an amplifiable region of a chromosome. - View Dependent Claims (53, 54, 55, 56, 57, 58, 59, 60, 61)
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62. A method for screening, comprising:
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contacting a cell comprising a reporter ACes with test compounds or known compounds, wherein;
the reporter ACes comprises one or a plurality of reporter constructs;
a reporter construct comprises a reporter gene in operative linkage with a regulatory region responsive to test or known compounds; and
detecting any increase or decrease in signal output from the reporter gene, wherein a change in the signal is indicative of activity of the test or known compound on the regulatory region. - View Dependent Claims (63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75)
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76. A reporter ACes comprises one or a plurality of reporter constructs, wherein the reporter construct comprises a reporter gene in operative linkage with a regulatory region responsive to test or known compounds.
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77. A method for obtaining site-specific recombination within a plant cell, comprising:
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introducing into the genome of a plant cell a nucleic acid molecule containing one or more recombinase recognition sites;
introducing into the plant cell an artificial chromosome containing a recombinase recognition site; and
introducing into the plant cell a gene encoding a recombinase enzyme, whereby plant chromosomal DNA from the genome of the plant cell is introduced into the artificial chromosome. - View Dependent Claims (78, 79, 80, 81, 82, 83, 84)
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85. A method of conferring a trait on a plant cell, comprising:
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introducing into a first plant cell a nucleic acid molecule containing one or more recombinase recognition sites;
introducing into the first plant cell an artificial chromosome containing a recombinase recognition site;
introducing into the first plant cell a gene encoding a recombinase enzyme, whereby plant chromosomal DNA from the genome of the plant cell is introduced into the artificial chromosome;
isolating the resulting artificial chromosome; and
introducing the isolated artificial chromosome into a second plant cell, whereby a trait from the first plant cell is conferred on the second plant cell.
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Specification