Y-Chromosome Detection in GFP CETN2 Mouse Tissues

Genomic DNA was isolated for Y-chromosome detection in GFP CETN2-expressing e11.5, e14.5 and e18.5 mouse tail tip tissues and PCR performed using MyTaq Extract-PCR Kit (Bioline, Taunton, MA). A 331-bp gene segment was amplified using the Jarid primers (forward: 5′ CTG AAG CTT TTG GCT TTG AG; reverse: 3′ CCG CTG CCA AAT TCT TTG G; Life Technologies, Carlsbad, CA). Β-actin loading control used the primers: 5′ GAT GAC GAT ATC GCT GCG CTG GTC G 3′ (forward), and 5′ GCC TGT GGT ACG ACC AGA GGC ATA CAG 3′ (reverse). PCR conditions were: 95 °C for 3 min, followed by 35 cycles of 95 °C for 15 seconds, 60 °C for 15 seconds, and 72 °C for 20 seconds. PCR product was analyzed in 2% agarose gel and visualized using ethidium bromide. Lanes expressing two bands were classified as males, the remainder female. Direct immunofluorescence of GFP CETN2 expression from non-gonadal cells at e11.5, e14.5, and e18.5 stages was accomplished as previously described^{25 (link)}.