Embryonic stem cells (ES) are derived from the inner cell mass of the mouse blastocyst and can be perpetuated in culture. The use of site-specific recombination systems from the phage P1 and from Saccharomyces cerevisiae has opened new avenues for a more sophisticated genetic engineering of mice. Embryonic fibroblasts should be used within a week when plated after inactivation. The ES cells should be split every second day. To avoid differentiation, it is important that the ES cells are trypsinized to get a single cell suspension. The neomycin gene is therefore inserted into one exon or it may replace some of the coding sequences, which have to be deleted to abolish the function of the gene to be targeted. Optimal targeting frequencies are often obtained with constructs, including more than 10 kb of homology. The length of DNA on each side of the Neo cassette should not be less than 1.5 kb. Increased targeting efficiencies have been observed when using isogenic DNA isolated from the 129SV genomic library, the mouse line from which most ES cell lines are derived. © 2006 Copyright © 2006 Elsevier Inc. All rights reserved.
Mansouri, A. (2006). Gene Targeting by Homologous Recombination in Embryonic Stem Cells. In Cell Biology, Four-Volume Set (Vol. 3, pp. 491–499). Elsevier Inc. https://doi.org/10.1016/B978-012164730-8/50180-5