Abstract
The inactivation of selected genes in the prokaryotic and eukaryotic genome is a powerful tool in studying their function both at the level of individual cells and in the context of a complete organism. The method of gene inactivation relies on the ability of virtually every cell type to exchange DNA sequences with a high degree of sequence similarity by homologous recombination. In general, the procedure involves the generation of a targeting construct that consists of a selectable marker gene flanked by DNA sequences largely identical to the chromosomal locus to be modified. On entry of the targeting construct into the cell, exhange of the flanking sequences with their chromosomal counterparts will result in the introduction of the marker gene into the chromosome, thereby disrupting the gene of interest. This procedure can be efficiently applied on mouse embryonic stem (ES) cells, which, on fusion to recipient blastocysts, give rise to chimeric mice that can transmit the disrupted allele to their offspring (1). Inbreeding of heterozygous animals will yield offspring carrying the disruption in both alleles. The phenotypic consequences of a complete gene knockout in the mouse germ line can provide pivotal information on the function of the gene in development and maintenance of the organism. Moreover, valuable mouse models have been generated for a number of recessive genetic disorders in humans.
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© 2001 Humana Press Inc., Totowa, NJ
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te Riele, H., Brouwers, C., Dekker, M. (2001). Generation of Double-Knockout Embryonic Stem Cells. In: Tymms, M.J., Kola, I. (eds) Gene Knockout Protocols. Methods in Molecular Biology, vol 158. Humana Press. https://doi.org/10.1385/1-59259-220-1:251
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DOI: https://doi.org/10.1385/1-59259-220-1:251
Publisher Name: Humana Press
Print ISBN: 978-0-89603-572-0
Online ISBN: 978-1-59259-220-3
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