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Animals All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals from the Institute for Laboratory Animal Research of the National Academy of Sciences. B6.Ytir mice kept in our colony (backcross generations N 30-35) that possess the B6 genetic background and the Y chromosome from M. musculus domesticus (from Tirano, Italy) were produced as previously described. B6.Ytir males were mated with B6 females, and the day of vaginal plug was taken to be 0.5 days postcoitum (dpc). Bilateral gonads were dissected from embryos at 11.5 up to 18.5 dpc and 30 and 60 days postpartum (dpp). Zfy PCR Analysis The chromosomal sex of the B6-Ytir embryos was determined by PCR amplification of the 600 base pair (bp) fragment of the Y-encoded gene Zfy. The PCR was performed using DNA isolated from the fetal tissues or from the tails at 30 and 60 dpp. The tissue was homogenized and incubated in lysis buffer (100 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 0.5% Tween 20, 0.5% NP-40, and 0.4 mg/ml proteinase K) at 55°C overnight, and proteinase K was inactivated by heating at 85°C for 10 minutes. Fifteen microliters of the lysate were diluted in 85 |xl of ddH2O.