Table 2.
Period Estimates for Different BAC Transgenic Lines
| Transgenic Line1 | +/+ | +/+ tg | Clock/+ | Clock/+ tg | Clock/Clock | Clock/Clock tg |
|---|---|---|---|---|---|---|
| BAC 54 (140 kb circular)2 | ||||||
| TG14 | N/A | N/A | 24.22 ± 0.183 (n = 5) | 23.08 ± 0.146 (n = 7) | 27.06 ± 0.314 (n = 7) | 23.27 ± 0.099 (n = 9) |
| TG36 | 23.48 ± 0.048 (n = 11) | 22.89 ± 0.051 (n = 10) | 24.18 ± 0.053 (n = 20) | 23.21 ± 0.047 (n = 20) | 27.36 ± 0.282 (n = 8) | 23.18 ± 0.082 (n = 14) |
| TG55 | 23.41 ± 0.091 (n = 10) | 22.92 ± 0.137 (n = 8) | 24.12 ± 0.206 (n = 8) | 22.77 ± 0.099 (n = 7) | N/A | N/A |
| TG60 | N/A | N/A | 23.91 ± 0.1 (n = 13) | 23.13 ± 0.122 (n = 6) | N/A | N/A |
| BAC 54 (100 kb linear)3 | ||||||
| TG80 | 23.44 ± 0.101 (n = 8) | 23.50 ± 0.07 (n = 5) | 23.92 ± 0.125 (n = 9) | 23.64 ± 0.083 (n = 4) | N/A | N/A |
| TG97 | N/A | N/A | 23.93 ± 0.04 (n = 4) | 23.67 ± 0.065 (n = 7) | N/A | N/A |
| BAC 52 (160 kb circular)4 | ||||||
| TG121 | 23.50 ± 0.142 (n = 4) | 23.66 ± 0.125 (n = 2) | 23.99 ± 0.11 (n = 13) | 23.96 ± 0.032 (n = 5) | 26.83 ± 0.40 (n = 2) | 26.87 ± 0.161 (n = 4) |
| TG126 | N/A | N/A | 23.45 ± 0.155 (n = 5) | 23.57 ± 0.125 (n = 4) | 26.87 ± 0.569 (n = 3) | 25.54 ± 0.103 (n = 3) |
Period estimates of BAC transgenic (tg) animals and their nontransgenic littermates. The rhythm of locomotor activity was recorded from a total of 266 F1 progeny from 8 transgenic lines. Free-running circadian periods were calculated for the 20-day interval during exposure to DD (days 1–20) by a χ2 periodogram (Sokolove and Bushel, 1978) (see text). 21 animals were excluded from analysis due to missing data or lack of a single dominant or significant circadian periodicity being detected. The Clock genotype was assigned in all but 5 cases, which were recombinant, by SSLP genotype (see text); 5 recombinant non-transgenic animals were included in the analysis on the basis of their behavioral phenotype. Values shown are the mean ± standard error, with the number of animals that were analyzed from each group indicated beneath. N/A, not applicable.
A Generalized Linear Models (GLM) Analysis of Variance (ANOVA) was performed on the data presented here (NCSS 6.0). Significant effects of the transgenic construct used (DF = 2; F = 34.5; p < 0.00001), the Clock genotype (DF = 2; F = 246; p < 0.00001), and the presence of the transgene (DF = 1; F = 97.7; p < 0.00001) were detected. In addition, significant construct by genotype (DF = 4; F = 12.8; p < 0.00001), construct by transgene presence (DF = 2; F = 58.7; p < 0.00001), genotype by transgene presence (DF = 2; F = 40.5; p < 0.00001) and three-way interactions (DF = 4; F = 17.2; p < 0.00001) were detected. This indicates that circadian period is affected by Clock genotype, by transgenic BAC DNA, and by the transgenic construct used. The effect of the transgene depends on the construct used (full-length BAC 54 had an effect; the others did not), and on the Clock genotype (i.e., greater period shortening by the transgene is seen in Clock mutants than in wild-type mice).
Among the lines involving this transgenic construct, a 3-way GLM ANOVA was performed. Significant effects of Clock genotype (DF = 2; F = 68.58; p < 0.0001), transgene presence (DF = 1; F = 149.67; p < 0.005), and genotype by transgene presence interaction (DF = 2; F = 60.34; p = 0.000106) were detected. This transgenic construct is effective in shortening period, and does so differentially with respect to the Clock genotype. No significant effect of transgenic line was detected (DF = 3; F = 1.8; p = 0.149). The effects of the BAC 54 transgene on circadian period thus apparently do not depend on the position of incorporation or level of transgene expression in this analysis.
Among the lines involving this transgenic construct, a 2-way GLM ANOVA was performed. Clock genotype (DF = 1; F = 13.1; p < 0.001) was significant, but no significant effect of the transgene presence (DF = 1; F = 1.41; p = 0.244) was detected. The 100 kb fragment of BAC 54 fails to shorten circadian period significantly as a transgene.
Among the lines involving this transgenic construct, a 2-way GLM ANOVA was performed. Clock genotype (DF = 1; F = 130; p < 0.00001) was significant, but no significant effect of the transgene presence (DF = 1; F = 0.61; p = 0.438) was detected. Transgenic BAC 52 fails to shorten circadian period significantly.