Method for genetically modifying isolated non-human mammalian cell by homologous recombination
First Claim
1. A method for targeting a cloned genomic fragment to a desired position in an isolated non-human mammalian cell genome, comprising(a) obtaining a vector comprising a donor fragment having a homology box 1 (hb1) of at least 40 nucleotides in length upstream and a homology box 2 (hb2) of at least 40 nucleotides in length downstream appended to a human genomic fragment containing the genomic sequence to be inserted into the genome of an isolated non-human mammalian cell;
- (b) obtaining a second vector comprising a target fragment isolated from a non-human mammalian cell, wherein the second vector accommodates DNA fragments greater than 20 kb;
(c) introducing both vectors into a bacterium;
(d) permitting bacterial homologous recombination between the donor fragment and the target fragment such that the human genomic fragment is inserted into a region to be modified in the non-human mammalian genomic target fragment, thereby producing a Large Targeting Vector for use in Eukaryotic Cells (LTVEC) comprising a modified non-human mammalian genomic fragment;
(e) introducing the LTVEC into the isolated non-human mammalian cell to target and modify an endogenous chromosomal locus in the isolated non-human mammalian cell, thereby forming a modified locus comprising the human DNA sequence resulting in a modified non-human mammalian cell; and
,(f) assaying the modified non-human mammalian cell for the modified locus.
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Abstract
A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification.
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Citations
54 Claims
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1. A method for targeting a cloned genomic fragment to a desired position in an isolated non-human mammalian cell genome, comprising
(a) obtaining a vector comprising a donor fragment having a homology box 1 (hb1) of at least 40 nucleotides in length upstream and a homology box 2 (hb2) of at least 40 nucleotides in length downstream appended to a human genomic fragment containing the genomic sequence to be inserted into the genome of an isolated non-human mammalian cell; -
(b) obtaining a second vector comprising a target fragment isolated from a non-human mammalian cell, wherein the second vector accommodates DNA fragments greater than 20 kb; (c) introducing both vectors into a bacterium; (d) permitting bacterial homologous recombination between the donor fragment and the target fragment such that the human genomic fragment is inserted into a region to be modified in the non-human mammalian genomic target fragment, thereby producing a Large Targeting Vector for use in Eukaryotic Cells (LTVEC) comprising a modified non-human mammalian genomic fragment; (e) introducing the LTVEC into the isolated non-human mammalian cell to target and modify an endogenous chromosomal locus in the isolated non-human mammalian cell, thereby forming a modified locus comprising the human DNA sequence resulting in a modified non-human mammalian cell; and
,(f) assaying the modified non-human mammalian cell for the modified locus. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A method for targeting a cloned genomic fragment to a desired position in an isolated non-human mammalian cell genome, comprising (a) obtaining a vector comprising a donor fragment having a homology box 1 (hb1) of at least 40 nucleotides in length upstream and a homology box 2 (hb2) of at least 40 nucleotides in length downstream appended to a human genomic fragment containing the genomic sequence to be inserted into the genome of an isolated non-human mammalian cell and orthologous to an endogenous chromosomal locus in the isolated non-human mammalian cell;
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(b) obtaining a second vector comprising a target fragment isolated from a non-human mammalian cell, wherein the second vector accommodates DNA fragments greater than 20 kb; (c) introducing both vectors into a bacterium; (d) permitting bacterial homologous recombination between the donor fragment and the target fragment such that the human genomic fragment is inserted into a region to be modified in the non-human mammalian genomic target fragment, thereby producing a Large Targeting Vector for use in Eukaryotic Cells (LTVEC) comprising a modified non-human mammalian genomic fragment; (e) introducing the LTVEC into the isolated non-human mammalian cell to target and modify the endogenous chromosomal locus in the isolated non-human mammalian cell, thereby forming a modified locus comprising the human DNA sequence resulting in a modified non-human mammalian cell; and
,(f) assaying the modified non-human mammalian cell for the modified locus. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
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45. A method for modifying a desired position in an isolated non-human mammalian cell genome, comprising
(a) obtaining a vector comprising a donor fragment having a homology box 1 (hb1) of at least 40 nucleotides in length upstream and a homology box 2 (hb2) of at least 40 nucleotides in length downstream appended to a DNA sequence to be inserted into the genome of an isolated non-human mammalian cell; -
(b) obtaining a second vector comprising a target fragment isolated from a non-human mammalian cell, wherein the second vector accommodates DNA fragments greater than 20 kb; (c) introducing both vectors into a bacterium; (d) permitting bacterial homologous recombination between the donor fragment and the target fragment such that the DNA sequence is inserted into a region to be modified in the non-human mammalian genomic target fragment, thereby producing a Large Targeting Vector for use in Eukaryotic Cells (LTVEC) comprising a modified non-human mammalian genomic fragment; (e) introducing the LTVEC into the isolated non-human mammalian cell to target and modify an endogenous chromosomal locus in the isolated non-human mammalian cell thereby forming a modified locus comprising the DNA sequence resulting in a modified non-human mammalian cell; and
,(f) assaying the modified non-human mammalian cell for the modified locus. - View Dependent Claims (46, 47, 48, 49, 50, 51, 52, 53, 54)
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Specification