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Nucleic Acids Research, 2000, Vol. 28, No. 21 e92
© 2000 Oxford University Press

Cre-mediated germline mosaicism: a method allowing rapid generation of several alleles of a target gene

Martin Holzenberger*, Claudia Lenzner1, Patricia Leneuve, Randa Zaoui, Ghislaine Hamard2, Sophie Vaulont1 and Yves Le Bouc

INSERM U515, Hôpital Saint-Antoine, 184 rue du Fbg St-Antoine, F-75571 Paris Cedex 12, France, 1INSERM U129 and 2U380, Faculté de Médecine Cochin-Port Royal, 24 rue Fbg St-Jacques, 75014 Paris, France

Conditional gene targeting uses the insertion of expression cassettes for the selection of targeted embryonic stem cells. The presence of these cassettes in the final targeted chromosomal locus may affect the normal expression of the targeted gene and produce interesting knock down phenotypes. We show here that the selection cassette may then be selectively removed in vivo, using three appropriately positioned loxP sites in the targeted gene and the transgenic mouse EIIaCre. This strategy was applied to two different target genes and we demonstrated that it is reliable and reproducible. First, we generated double transgenic EIIaCre/loxP mice (F1) that showed variable degrees of mosaicism for partially Cre-recombined floxed alleles. Efficiency of EIIaCre at creating mosaicism was dependent on the target gene and on parental transmission of the transgene. The segregation of partially recombined alleles and EIIaCre transgene was obtained in the next generation using mosaic F1 males. Mosaic females were unsuitable for this purpose because they systematically generated complete excisions during oogenesis. Our strategy is applicable to other approaches based on three loxP sites. As this procedure allows generation of knock down (presence of neo), knockout (total exision of the loxP-flanked sequences) and floxed substrains (excision of the selection cassette) from a single, targeted germline mutation and in a single experiment, its use may become more widespread in conditional mutagenesis.

* To whom correspondence should be addressed. Tel: +33 1 49 28 46 29; Fax: +33 1 43 43 10 65; Email: holzenberger@st-antoine.inserm.fr


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