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Method for genetically modifying isolated non-human mammalian cell by homologous recombination

  • US 8,846,402 B2
  • Filed: 12/19/2012
  • Issued: 09/30/2014
  • Est. Priority Date: 10/31/2000
  • Status: Expired due to Fees
First Claim
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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.

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