Direct removal in the mouse of a flexed neo gene from a three-loxP conditional knockout allele by two novel approaches

The presence in an intron of the ploxP-neo-loxP cassette often results in severe interference with gene expression. Consequently, many investigators selectively remove the ploxP-neo-loxP cassette by transient expression of Cre in ES cells, Although effective, the added manipulation of the ES cells may reduce the likelihood that a clone will be able to transmit via the germline, Therefore, we developed two novel approaches that remove the ploxP-neo-loxP by Cre-mediated recombination in mouse. First, the ploxP-neo-loxP-containing mice were crossed with Ella-Cre transgenic mice. Second, a Cre-expression plasmid was injected into pronuclei of fertilized eggs bearing the ploxP-neo-loxP allele, Both approaches produced mosaic mice with partial and complete excision. These mosaic mice were then mated, and the neo-less conditional knockout allele was found in the offspring after screening only a few litters. These procedures provide options for removing neo directly in the mouse in addition to the commonly used approach that deletes neo in ES cells. genesis 30: 1-6, 2001, Published 2001 Wiley-Liss, Inc.(dagger)