Genotyping
A DNA identification procedure was performed in order to evaluate the
reliability of COX-2KO transgenic mice. Approximately 4 mm was cut from
the tails of mice. Total DNA was isolated from the mouse with a DNA
extraction kit (Beijing Tiangen Biochemical Technology, China) and was
amplified by PCR. The following conditions were applied to the PCR
reaction system: initial denaturation for 4 minutes at 94 °C, followed
by 31 cycles of denaturation for 30 seconds at 94 °C, annealing for 30
seconds at 61 °C, and extension for 1 minute at 72 °C, followed by a
final extension for 5 minutes at 72 °C. A gel imaging device (Bio-Rad,
USA) is used to visualize the PCR products after they have been labeled
with SYBR Green (Sigma-Aldrich, USA), separated by electrophoresis (130V
constant voltage, continued for 30 minutes) and visualized with SYBR
Green (Sigma-Aldrich, USA). The following primers (Sangon Biotech,
China) were used: CDF141: 5’-ATC GCC TTC TTG ACG AGT TC-3’; COX2KO
probe1: 5’- ACC TCT GCG ATG CTC TTCC-3’; COX2KO probe2: 5’- ACT GGT CAA
ATC CTG TGC TC-3’.