EFFICIENT GENE TARGETING IN MOUSE EMBRYONIC STEM-CELLS

We developed methods to improve the efficiency of gene correction in m ouse embryonic stem cells using homologous recombination of a replacem ent vector. The absolute frequency of homologous recombination in mous e embryonic stem (ES) cells, defined as the frequency of homologous re combination per electroporated cell, is approximately 10(-5) to 10(-6) by current procedures. Our method for gene targeting in mouse ES cell s 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 elec troporated cells at an optimum density to maintain viability significa ntly increased the recovery of targeted cells. Due to the high frequen cy of targeting, corrected cells could be isolated by screening coloni es obtained after growth without selection. Alternatively, colony form ation and the absolute cells with nonelectroporated ES cells before th e addition of selective medium. These parental cells were nonirradiate d but were killed in the selective medium. Plating density and efficie ncy of colony formation are therefore of homologous recombination. Bec ause this frequency is extremely high, these methods can be used to pe rform gene targeting without the use of selectable markers.