Alteration of the mouse genome through homologous recombination in embryonic stem (ES) cells is the most accurate and versatile way to dissect gene function in a vertebrate model. Most often, a selectable marker is used to create a knockout allele by replacing an essential part of the gene. However, knockout strategies are limited because the mutation is present constitutively. Conditional approaches based on the Cre-loxP site-specific recombination (SSR) system address this limitation; however, it requires that all parts of the targeted gene remain in ES cells. Here we report success with a "knockout-first" strategy that ablates gene function by insertion of RNA processing signals without deletion of any of the target gene. Incorporation of site-specific recombination target sites creates a multipurpose allele for both knockout and conditional applications.
A Reliable lacZ Expression Reporter Cassette for Multipurpose, Knockout-First Alleles / G. Testa, J. Schaft, F. Van Der Hoeven, S. Glaser, K. Anastassiadis, Y. Zhang, T. Hermann, W. Stremmel, A.F. Stewart. - In: GENESIS. - ISSN 1526-954X. - 38:3(2004), pp. 151-158.
A Reliable lacZ Expression Reporter Cassette for Multipurpose, Knockout-First Alleles
G. Testa;
2004
Abstract
Alteration of the mouse genome through homologous recombination in embryonic stem (ES) cells is the most accurate and versatile way to dissect gene function in a vertebrate model. Most often, a selectable marker is used to create a knockout allele by replacing an essential part of the gene. However, knockout strategies are limited because the mutation is present constitutively. Conditional approaches based on the Cre-loxP site-specific recombination (SSR) system address this limitation; however, it requires that all parts of the targeted gene remain in ES cells. Here we report success with a "knockout-first" strategy that ablates gene function by insertion of RNA processing signals without deletion of any of the target gene. Incorporation of site-specific recombination target sites creates a multipurpose allele for both knockout and conditional applications.File | Dimensione | Formato | |
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