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Efficient Gene Targeting in Mouse Embryonic Stem Cells

  1. Author:
    Templeton, N. S.
    Roberts, D. D.
    Safer, B.

  2. Author Address

    Templeton NS NCI FREDERICK CANC RES & DEV CTR ABL BASIC RES PROGRAM POB B BOYLES ST BLDG 535 ROOM 226A FREDERICK, MD 21702 USA NCI PATHOL LAB NIH BETHESDA, MD 20892 USA NHLBI MOL HEMATOL BRANCH NIH BETHESDA, MD 20892 USA
    1. Year: 1997
  2. Journal: Gene Therapy
    1. 4
    2. 7
    3. Pages: 700-709
  4. Type of Article: Article
  1. Abstract:

    We developed methods to improve the efficiency of gene correction in mouse embryonic stem cells using homologous recombination of a replacement vector. The absolute frequency of homologous recombination in mouse embryonic stem (ES) cells, defined as the frequency of homologous recombination per electroporated cell, is approximately 10(-5) to 10(-6) by current procedures. Our method for gene targeting in mouse ES cells procedures an absolute frequency of 10(-1). The protocol uses micro-electroporation chambers and a modified electroporation procedure that does not cause significant cell death. Plating and growth of the electroporated cells at an optimum density to maintain viability significantly increased the recovery of targeted cells. Due to the high frequency of targeting, corrected cells could be isolated by screening colonies obtained after growth without selection. Alternatively, colony formation and the absolute cells with nonelectroporated ES cells before the addition of selective medium. These parental cells were nonirradiated but were killed in the selective medium. Plating density and efficiency of colony formation are therefore of homologous recombination. Because this frequency is extremely high, these methods can be used to perform gene targeting without the use of selectable markers. [References: 30]

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