Efficient gene targeting in mouse embryonic stem cells

• Published in: Gene therapy Vol. 4; no. 7; pp. 700 - 709
• Main Authors: TEMPLETON, N. S, ROBERTS, D. D, SAFER, B
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing Group 01.07.1997
• Subjects: Animals; Biological and medical sciences; Biotechnology; Blotting, Southern; Cell death; Cells, Cultured; Colonies; Electroporation; Embryo cells; Fundamental and applied biological sciences. Psychology; Gene targeting; Gene Targeting - methods; Gene therapy; Health. Pharmaceutical industry; Homologous recombination; Hypoxanthine Phosphoribosyltransferase - analysis; Hypoxanthine Phosphoribosyltransferase - genetics; Industrial applications and implications. Economical aspects; Mice; Recombination, Genetic; Selective media; Stem cell transplantation; Stem Cells; Embryo; Targeting; Stem cell; Homologous recombination; Glycosyltransferases; Hypoxanthine phosphoribosyltransferase; Enzymopathy; Hyperuricemia; Gene; Nucleic acid; Nervous system diseases; Enzyme; Transferases; Deficiency; Rodentia; Metabolic diseases; Electroporation; Lesch Nyhan syndrome; Method; Genetic disease; Genetic transfer; Vertebrata; Mammalia; Mouse; Gene therapy; Pentosyltransferases
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/sj.gt.3300457

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 produces 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 frequency could be increased by co-plating the electroporated 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 critical factors for obtaining a high absolute frequency of homologous recombination. Because this frequency is extremely high, these methods can be used to perform gene targeting without the use of selectable markers.