Mansour S L, Thomas K R, Deng C X, Capecchi M R
Howard Hughes Medical Institute, Department of Biology, University of Utah, Salt Lake City 84112.
Proc Natl Acad Sci U S A. 1990 Oct;87(19):7688-92. doi: 10.1073/pnas.87.19.7688.
We demonstrate that the frequency of gene targeting is unaffected by the length of nonhomologous DNA transferred to a target chromosomal sequence. A result of this finding is that a much wider spectrum of designed genomic alterations is now feasible. As a first application, we inserted a 5.4-kilobase cassette of nonhomologous DNA into the int-2 locus in mouse embryo-derived stem cells by gene targeting. The inserted DNA contained a lacZ gene positioned to create an in-frame fusion with the int-2 protein-coding region. Upon differentiation of these cells to embryoid bodies, the int-2-lacZ fusion faithfully reproduced the expression pattern of int-2 RNA. This ability to target reporter genes, such as lacZ, to specific mouse loci, combined with the ability to move the tagged gene into different mutant backgrounds, may provide an ideal approach for analyzing interactions among genes that participate in a developmental network.
