Direct removal in the mouse of a floxed neo gene from a three‐loxp conditional knockout allele by two novel approaches

Abstract

Summary: 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 EIIa‐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.