Table 1. Knockdown of Tyr in vivo is not sufficient to induce significant melanin loss in the black mouse coat.
| Outcome | Control (n = 5) | rtTA3/Tyr-shRNA (n = 5) | Difference (rtTA3/Tyr-shRNA–Cont) | % of Control | |
|---|---|---|---|---|---|
| Mean (SD) | Mean (SD) | Diff in Means (95% CI) | p-value | ||
| A500/mg | 0.76 (0.09) | 0.71 (0.08) | -0.06 (-0.18, 0.06) | 0.332 | |
| Total melanin (μg/mg) | 81.08 (10.65) | 73 (3.72) | -8.08 (-19.5, 3.34) | 0.148 | 90.1% |
| PCTA (ng/mg) | 3234.6 (423.9) | 2915.6 (148.9) | -319 (-773.6, 135.6) | 0.151 | |
| Eumelanin (μg/mg) | 80.88 (10.6) | 72.9 (3.71) | -7.98 (-19.34, 3.38) | 0.151 | 90.1% |
| 4-AHP (ng/mg) | 23.44 (7.4) | 14.04 (6.88) | -9.4 (-19.63, 0.83) | 0.071 | |
| Pheomelanin (μg/mg) | 0.22 (0.084) | 0.14 (0.055) | -0.08 (-0.18, 0.02) | 0.111 | 63.6% |
| Average Phe/Eu Ratio | 0.003 (0.001) | 0.002 (0.001) | 0.001 (-0.001, 0.001) | 0.185 |
Dorsal hairs of non-agouti double transgenic and control mice on continuous doxycycline treatment were shaved and processed for chemical analysis of eumelanin and of pheomelanin based upon their specific degradation products, pyrrole-2,3,5-tricarboxylic acid (PCTA) and 4-amino-3-hydroxyphenylalanine (4-AHP), respectively. Approximately 20 mg of hair was homogenized with a Ten Broeck homogenizer at a concentration of 10 mg/mL and 100 μL aliquots were subjected to Soluene-350 solubilization to quantify total melanin, alkaline hydrogen peroxide oxidation to quantify total PCTA, and hydroiodic acid hydrolysis to quantify 4-AHP in duplicate. The data corresponds to the averages of five control mice (Tyr-shRNA only or rtTA driver only) and five Tyr-knockdown mice, and the value is the mean of duplicate measurements. Each hair sample was derived from a single mouse. Within group summary statistics represent the arithmetic mean and (standard deviation). Differences in means between rtTA3/Tyr-shRNA and control were tested using a two-sample t-test with no assumption of equal variances between groups. P-values correspond to a test of the null hypothesis of no difference in true means between the groups. Percent of control is presented for descriptive purposes and represents the relative mean for rtTA3/Tyr-shRNA when compared to control. See also S2 Fig.