Direct removal in the mouse of a floxed 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 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.
Xu
X
X
Genetics of Development and Disease Branch, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, Maryland, USA.
Li
C
C
Garrett-Beal
L
L
Larson
D
D
Wynshaw-Boris
A
A
Deng
C X
CX
D000483:Alleles; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003433:Crosses, Genetic; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005091:Exons; D005260:Female; D018014:Gene Transfer Techniques; D019398:Genes, BRCA1 / Q000235:genetics; D005822:Genetic Vectors; D006579:Heterozygote; D019426:Integrases / Q000235:genetics*; D007438:Introns; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008957:Models, Genetic; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology; D014764:Viral Proteins / Q000235:genetics*
D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases