Nucleic Acids Research, Vol 25, Issue 12 2381-2388, Copyright © 1997 by Oxford University Press
LM Whyatt and PD Rathjen
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 integration of multiple regions of extended
non-homology, and providing an efficient system for the repeated
introduction of precise alterations into the mammalian genome.
ARTICLES
Introduction of precise alterations into the mouse genome with high efficiency by stable tag-exchange gene targeting: implications for gene targeting in ES cells
Department of Biochemistry, The University of Adelaide, Adelaide, SA 5005, Australia.
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