Introduction of precise alterations into the mouse genome with high efficiency by stable tag-exchange gene targeting: implications for gene targeting in ES cells

• Published in: Nucleic acids research Vol. 25; no. 12; pp. 2381 - 2388
• Main Authors: Whyatt, Linda M., Rathjen, Peter D.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 15.06.1997
• Subjects: Animals; Cell Line; Gene Transfer Techniques; Genes, Reporter; Genetic Vectors; Genome; Growth Inhibitors - biosynthesis; Growth Inhibitors - genetics; Hypoxanthine Phosphoribosyltransferase - biosynthesis; Hypoxanthine Phosphoribosyltransferase - genetics; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines - biosynthesis; Lymphokines - genetics; Mammals; Mice; Mice, Transgenic; Recombination, Genetic; Restriction Mapping; Stem Cells - cytology; Stem Cells - physiology
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/25.12.2381

The efficiency of tag-and-exchange gene targeting approaches for the introduction of precise genomic modifications is compromised by high levels of nonhomologous recombinants which survive selection due to loss of tag gene expression, often by physical loss of the tag gene. We describe a modified approach, termed stable tag-exchange, which incorporates an additional positive selection (stability) cassette to circumvent this limitation. HPRT (tag) and neo (stability) cassettes, separated by 4.9 kb of homologous DNA, were introduced efficiently into the LIF locus of ES cells. The tag cassette was substituted for a β-galactosidase gene in exchange step targeting. Direct comparison of the tag-and-exchange and stable tagexchange approaches indicated respective targeting efficiencies of 21% and 88%. The increased stable tag-exchange targeting efficiency resulted from elimination of >75% of background lines which survived tag-and-exchange selection due to physical loss of the tag gene. These resulted from reversion of the tagged allele to wild-type which is therefore a major contributor to tag-and-exchange targeting background. Our results extend the application of gene targeting by demonstrating a rationale for single-step integration of multiple regions of extended non-homology, and providing an efficient system for the repeated introduction of precise alterations into the mammalian genome.