The improved method of homologous recombination in eukaryotic cell

The improved method of homologous recombination in eukaryotic cell

  • CN 101,421,401 A
  • Filed: 03/14/2007
  • Published: 04/29/2009
  • Est. Priority Date: 04/08/2006
  • Status: Active Application
First Claim
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1. make up eukaryotic method, the target sequence in the described eukaryotic cell chromosomal DNA sequence is replaced into the constant series of wanting, and described method comprises:

  • Modification provides the eukaryotic cell of comparing the HR/NHR ratio with raising with described parental cell to parent'"'"'s eukaryotic cell that NHR has preference,The dna molecular that comprises first dna fragmentation and second dna fragmentation is provided, described first dna fragmentation comprises the constant series of wanting, the flank of these constant series 5 '"'"' and 3 '"'"' side is and the basic homologous dna sequence dna of the chromosomal DNA sequence of target sequence flank, described second dna fragmentation comprises expression cassette, the adjusting sequence that described expression cassette comprises the gene of the selectable marker of encoding and is operably connected with it, function is arranged in eukaryotic cell;

    Transform described modified eukaryotic cell with described dna molecular;

    Cultivate described cell, obtain progeny cell, inserted described dna molecular in the described progeny cell karyomit(e) through transforming,By expressing described second marker, the selected wherein said dna molecular of cancellation is inserted into progeny cell in the described karyomit(e) by non-homogeneous recombination event;

    WithObtain described target sequence in the wherein said chromosomal DNA sequence by the described constant series metathetical cell of wanting.

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