Transposon-mediated BAC transgenesis in human ES cells.

Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes.

An improved Flp deleter mouse in C57Bl/6 based on Flpo recombinase.

Recently, a codon improved version of the Flpe site specific recombinase, termed Flpo, was reported as having greatly improved performance in mammalian cell applications. However, the degree of improvement could not be estimated because essentially no Flpe activity was observed. Here, we compare Flpe and Flpo accurately in a mammalian cell assay to estimate that Flpo is about five times more active than Flpe and similar to Cre and Dre.

Precise conditional immortalization of mouse cells using tetracycline-regulated SV40 large T-antigen.

Cellular immortalization provides a way for expansion and subsequent molecular characterization of rare cell types. Ideally, immortalization can be achieved by the reversible expression of immortalizing proteins. Here, we describe the use of conditional immortalization based on a modified tetracycline-regulated system for the expression of SV40 large T-antigen in embryonic stem (ES) cells and mice.

Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice.

Tyrosine site-specific recombinases (SSRs) including Cre and FLP are essential tools for DNA and genome engineering. Cre has long been recognized as the best SSR for genome engineering, particularly in mice. Obtaining another SSR that is as good as Cre will be a valuable addition to the genomic toolbox.