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Foxn1[Cre] Expression in the Male Germline

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Recombined alleles appear independently of Cre expression in offspring from Foxn1[Cre]+ male, but not female, mice.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.
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pone.0166967.g001: Recombined alleles appear independently of Cre expression in offspring from Foxn1[Cre]+ male, but not female, mice.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.

Mentions: During a study where Foxn1-driven Cre recombinase [5] was used to delete a loxP-flanked Birc5 gene [4] in thymic epithelial stromal cells, we made the observation that Cre-negative offspring unexpectedly exhibited the presence of rearranged Birc5fl alleles. We established colony specifically to further test this observation (Fig 1). We found that offspring derived from female Foxn1[Cre] mice (crossed to C57BL/6 males) were genotyped in the expected ratios, exhibiting a recombined Birc5 allele only when Foxn1[Cre] has also been inherited (note: DNA from skin, which was the tissue used as a template for genotyping, includes both keratinocytes that express Foxn1 and other cell types that do not, and thus both a recombined allele and a floxed allele are detected when Cre is expressed). This was not the case, however, when male Foxn1[Cre] mice were crossed to C57BL/6 females. Instead, all offspring exhibited a recombined loxP allele, independent of whether they also inherited Cre. Importantly, even in Foxn1 non-expressing or mixed tissues (such as skin), a non-recombined allele was absent, suggesting that recombination of the floxed allele had occurred not only independent of transmission of Foxn1[Cre], but also prior to divergence of epithelial and mesenchymal (or other) tissue lineages.


Foxn1[Cre] Expression in the Male Germline
Recombined alleles appear independently of Cre expression in offspring from Foxn1[Cre]+ male, but not female, mice.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120802&req=5

pone.0166967.g001: Recombined alleles appear independently of Cre expression in offspring from Foxn1[Cre]+ male, but not female, mice.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.
Mentions: During a study where Foxn1-driven Cre recombinase [5] was used to delete a loxP-flanked Birc5 gene [4] in thymic epithelial stromal cells, we made the observation that Cre-negative offspring unexpectedly exhibited the presence of rearranged Birc5fl alleles. We established colony specifically to further test this observation (Fig 1). We found that offspring derived from female Foxn1[Cre] mice (crossed to C57BL/6 males) were genotyped in the expected ratios, exhibiting a recombined Birc5 allele only when Foxn1[Cre] has also been inherited (note: DNA from skin, which was the tissue used as a template for genotyping, includes both keratinocytes that express Foxn1 and other cell types that do not, and thus both a recombined allele and a floxed allele are detected when Cre is expressed). This was not the case, however, when male Foxn1[Cre] mice were crossed to C57BL/6 females. Instead, all offspring exhibited a recombined loxP allele, independent of whether they also inherited Cre. Importantly, even in Foxn1 non-expressing or mixed tissues (such as skin), a non-recombined allele was absent, suggesting that recombination of the floxed allele had occurred not only independent of transmission of Foxn1[Cre], but also prior to divergence of epithelial and mesenchymal (or other) tissue lineages.

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus