PMID: 9171089Jun 15, 1997Paper

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

Nucleic Acids Research
L M Whyatt, P D Rathjen

Abstract

The efficiency of tag-and-exchange gene targeting approaches for the introduction of precise genomic modifications is compromised by high levels of non-homologous 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 abeta-galactosidase gene in exchange step targeting. Direct comparison of the tag-and-exchange and stable tag-exchange 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 integr...Continue Reading

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Citations

Aug 1, 1998·Mutation Research·R P WoychikE D Avrer
Aug 6, 2013·Genesis : the Journal of Genetics and Development·Douglas B Menke
Dec 5, 2002·Physiological Genomics·Louise van der WeydenAllan Bradley
Jan 30, 2010·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Ingo D MeierMelitta Schachner

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