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First published online December 14, 2007
Journal of Experimental Biology 211, 128-137 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.006890

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Myogenesis and muscle metabolism in juvenile Atlantic salmon (Salmo salar) made transgenic for growth hormone

H. M. Levesque^1,*, M. A. Shears², G. L. Fletcher² and T. W. Moon^1,

¹ Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, PO Box 450, Stn A, Ottawa, Ontario, Canada, K1N 6N5
² Ocean Sciences Centre, Memorial University of Newfoundland, and AquaBounty Technologies Inc., St John's, Newfoundland, Canada, A1C 5S7

View larger version (6K): [in this window] [in a new window]   Fig. 1. PCR analysis to detect the presence of the growth hormone (GH) transgene from fin clips of GH-transgenic and non-transgenic Atlantic salmon (Salmo salar). The PCR conditions were 92°C for 30 s, 60°C for 30 s and 72°C for 30 s, for 32 cycles. The primers anneal to the Chinook salmon GH cDNA within the EO-1 transgene producing a 207 bp amplicon. In addition, the primers also anneal within the endogenous Atlantic salmon GH DNA sequence, producing amplicons of 798 bp and 1150 bp that serve as internal positive controls for the PCR reaction. Lane 1, DNA ladder; lanes 2 and 3, fin clip from GH-transgenic salmon; lanes 4 and 5, fin clip from non-transgenic salmon; lane 6, negative control (H2O); lane 7, positive control.

View larger version (7K): [in this window] [in a new window]   Fig. 2. White muscle myosatellite cell (MC) proliferation in vitro. •, MCs extracted from 4 month old GH-transgenic salmon; , MCs extracted from 4 month old non-transgenic salmon; , MCs extracted from 7 month old non-transgenic salmon. Sample size equals 3 for transgenic and non-transgenic salmon at 4 months (control age) and 5 for non-transgenic salmon at 7 months (control size). Results are means ± s.d.; a two way ANOVA was performed using the effects of days after plating (P<0.001), presence of transgene (P<0.001) and the interaction (P=0.0029); asterisk indicates significant differences between transgenic and non-transgenic fish at 7 months (Bonferroni, P<0.05).

View larger version (5K): [in this window] [in a new window]   Fig. 3. Non-transgenic salmon white muscle MC proliferation in vitro exposed to GH at 10 ng l–1 (GH10) and 50 ng l–1 (GH50). Sample size equals 3 for each group. Results are means ± s.d.; asterisk indicates significant difference from control (one-way ANOVA, P=0.00072, Tukey–Kramer, <0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 4. (A) Relative expression of MyoD I mRNA in white muscle of GH-transgenic and non-transgenic Atlantic salmon at 4 and 7 months of age. Sample size equals 11 for non-transgenic salmon at 4 months, 12 for transgenic salmon at 4 months, 9 for non-transgenic salmon at 7 months and 9 for transgenic salmon at 7 months. Results are means ± s.e.m. Data were log transformed to obtain normality. Two-way ANOVA was used, assessing the effect of age (P<0.001), presence of the transgene (P=0.452) and the interaction of age and the transgene (P=0.790). Different letters and horizontal bars indicate significant differences between ages of the fish. Relative expression is the ratio of the band intensity of the gene of interest to that of the comparator gene, 18S. (B) Picture of the gel used to analyse band intensities.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Relative expression of myogenin mRNA in white muscle of GH-transgenic and non-transgenic Atlantic salmon at 4 and 7 months of age. Sample size equals 11 for non-transgenic salmon at 4 months, 12 for transgenic salmon at 4 months, 9 for non-transgenic salmon at 7 months and 9 for transgenic salmon at 7 months. Results are means ± s.e.m. Different letters indicate significant differences. Myogenin data were log transformed to obtain normality. Two-way ANOVA was used, assessing the effect of age (P<0.001), presence of the transgene (P=0.528) and the interaction of age and the transgene (P=0.002). See Fig. 4 legend for the definition of relative expression. (B) Picture of the gel used to analyse band intensities.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Relative expression of (A) MyoD I and (B) MyoD II mRNA in red muscle of GH-transgenic and non-transgenic Atlantic salmon at 4 and 7 months of age. Sample size equals 11 for transgenic salmon at 4 months, 10 for non-transgenic salmon at 7 months and 10 for transgenic salmon at 7 months. Results are means ± s.e.m. MyoD I data were log transformed to obtain normality. Data were analysed using one-way ANOVA, P=0.006 for MyoD I and P<0.001 for MyoD II (Tukey–Kramer, <0.05). Red muscle could not be sampled from control salmon at 4 months due to the small size of the fish. Different letters indicate significant differences. See Fig. 4 legend for the definition of relative expression. (C) Picture of the gel used to analyse band intensities.

View larger version (12K): [in this window] [in a new window]   Fig. 7. Relative expression of myogenin RNA expression in red muscle of GH-transgenic and non-transgenic Atlantic salmon at 4 and 7 months of age. Sample size equals 11 for transgenic salmon at 4 months, 10 for non-transgenic salmon at 7 months and 10 for transgenic salmon at 7 months. Results are means ± s.e.m. Myogenin data were log transformed to obtain normality. Data were analysed using one-way ANOVA, P=0.001 (Tukey–Kramer, <0.05). Red muscle could not be sampled from control salmon at 4 months due to the small size of the fish. Different letters indicate significant differences. See Fig. 4 legend for the definition of relative expression. (B) Picture of the gel used to analyse band intensities.

View larger version (7K): [in this window] [in a new window]   Fig. 8. Pyruvate kinase (PK) activity in GH-transgenic and non-transgenic salmon white muscle. Sample size equals 8 or more for each group. Results are means ± s.e.m. PK data were r2 transformed to obtain normality. Two-way ANOVA was used, assessing the effect of age (P<0.001), presence of the transgene (P<0.001) and the interaction of age and the transgene (P=0.019). Pearson correlations between mass and white muscle PK equal 0.906, P<0.001 (Bonferroni). Different letters indicate significant differences.

View larger version (6K): [in this window] [in a new window]   Fig. 9. Citrate synthase (CS) activity in GH-transgenic and non-transgenic salmon liver. Sample size equals 8 or more for each group. Results are means ± s.e.m. Two-way ANOVA was used, assessing the effect of age (P<0.001), presence of the transgene (P<0.001) and the interaction of age and the transgene (P<0.001). Pearson correlations between mass and liver CS equal 0.905, P<0.001 (Bonferroni). Different letters indicate significant differences.