Foxn1[Cre] Expression in the Male Germline.

Foxn1 (forkhead box N1), also known as the nude gene or winged-helix nude (Whn), is a forkhead transcription factor thought to be restricted to keratinocytes in the skin and thymus. Consistent with this tissue distribution, spontaneous or targeted mutation of Foxn1 results in the absence of both hair and a thymus. Genetic manipulation of the Foxn1 locus thus represents a powerful tool for tissue specific gene control in the skin and thymus, and tools such as Cre recombinase under control of the Foxn1 locus are widely used for this purpose. Unexpectedly, we show that Foxn1[Cre] exhibits unexpected activity in male germ cells, resulting in ubiquitous targeting of loxP-flanked alleles in all tissues in offspring from Foxn1[Cre] expressing male mice. Inheritance of recombined loxP alleles occurs independently of Cre inheritance (i.e., offspring lacking Cre nonetheless exhibit recombined alleles), suggesting that Foxn1[Cre] induced recombination in male germ cells must occur prior to meiosis in diploid germ cells. Together with previously published data, our results show that Foxn1, and alleles under its control, are expressed in the pre-meiotic male germline, revealing a new tool for germline targeting of genes, and raising important concerns for gender selection when using Foxn1 regulatory elements

Inappropriate reporter activity, independent of Cre expression, in offspring from Cre+ male, but not female, mice.

<p>a) a conditional Gfp reporter expression in white blood cells (WBC) from Cre+ or Cre- offspring of Cre+ male or female parents (x B6 cross). WBC are uniformly Gfp+ when the male parent was Cre+, regardless of whether the offspring inherited Cre. Expression of a Foxn1[Cre]-activated conditional reporter is not expected in WBC from either Cre+ or Cre- offspring, since Foxn1 is not expressed in blood cells, as is the case when Foxn1[Cre] was carried by the mother. b) a summary of larger numbers of offspring from multiple independent litters.</p

Recombined alleles appear independently of Cre expression in offspring from Foxn1[Cre]+ male, but not female, mice.

<p>a) PCR gel images using primers specific for Cre (top), primers that distinguish loxP-flanked (fl/fl) from wildtype (wt) alleles (middle), or primers that detect a recombined Birc5 allele (bottom), using DNA template from skin. When the female parent carried both Cre and the Birc5 conditional allele, only Cre+ offspring exhibited a recombined allele, as expected. However, offspring of the equivalent male parent showed recombination of Birc5 even in the absence of Cre. b) a summary of offspring from multiple independent litters.</p