Tissue specific expression of antifreeze protein and growth hormone transgenes driven by the ocean pout (Macrozoarces americanus) antifreeze protein OP5a gene promoter in Atlantic salmon (Salmo salar)

Abstract

Previous research aimed at producing genetically improved salmon broodstock for aquaculture led to the creation of two lines of transgenic Atlantic salmon using gene constructs that were derived in part from the ocean pout OP5a antifreeze protein (AFP) gene. One of the lines was produced using an OP5a AFP gene in which the 5 0 region of the promoter was removed (termed t-OP5a-AFP), and the other line contains a growth hormone (GH) transgene (EO-1a) that consists of a chinook salmon GH cDNA driven by a truncated OP5a AFP promoter that is almost identical to that of the t-OP5a-AFP construct. The similarity of the promoter regions of these transgenes provided an opportunity to evaluate their tissue specific expression patterns. Expression of mRNA was evaluated using Northern blot and RT-PCR techniques. The results demonstrate that the AFP and GH trangenes were expressed in almost all body tissues, suggesting that the promoter region of the OP5a AFP gene lacks tissue specific elements. Northern analysis revealed that expression of the t-OP5a-AFP gene was considerably greater than that of the EO-1a GH transgene. Only the spleen tissue of the GH transgenics showed a visible band of hybridization. In contrast clear bands of hybridization were evident in all tissues, except for blood cells, of the AFP transgenics with heart, liver and brain tissue showing the highest levels of mRNA expression. This higher level of expression could be attributable to the presence of introns in the t-OP5a-AFP transgene. Since the GH transgenic salmon grow considerably faster than non-transgenics the low levels of GH transgene expression in this line were clearly sufficient to produce the desired rapid growth phenotype. In contrast the levels of AFP expression were inadequate to impart any improvement in the freeze resistance of the AFP transgenic salmon.

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

“…The GH-enhanced proliferation of white muscle MCs in vitro certainly indicates that GH can have a direct effect on these MCs as reported by Halevy et al (Halevy et al, 1996) and Hodik et al (Hodik et al, 1997) in avian muscle and Michal et al (Michal et al, 2002) in canine satellite cells. Although the levels of GH in the plasma of the transgenic salmon have not been quantified, GH transgene mRNA is expressed in most tissues including muscle of these transgenic Atlantic salmon (Hew et al, 1995;Hobbs and Fletcher, 2007). Therefore, it is possible that GH production by the muscle tissue and/or higher levels of circulating GH is responsible for the increased proliferation directly or via IGF-I production in muscle as reported for mice (Kim et al, 2005) and the C2C12 cell line (Sadowski et al, 2001).…”

Lysozyme transgenic Atlantic salmon (Salmo salar L.)

“…In contrast to the RT‐PCR results, Northern blot analysis revealed considerable differences between the transgenes with regard to tissues exhibiting the highest levels of expression. Antifreeze protein mRNA expression was evident in all body tissues while lysozyme mRNA was only evident in spleen, gill and kidney tissues and GH transgene expression in non‐pituitary tissues was confined to the spleen (Hobbs & Fletcher 2008).…”

“…Because RT‐PCR analyses revealed that all three transgenes were expressed in most body tissues, it is possible that the number (in parentheses) of tissues showing visible levels of transgene mRNA in GH (1), lysozyme (3) and AFP transgenics (12) is related to promoter strength. However, an additional factor that has to be taken into consideration is the presence of an intron in the AFP transgene (Hobbs & Fletcher 2008). Neither the GH nor the lysozyme transgenes contain introns.…”

Antifreeze protein gene amplification facilitated niche exploitation and speciation in wolffish

“…This control housekeeping gene transcript was detected in all tissues, but was particularly abundant in the brain, which is known to be rich in tubulin [57,58]. The signal was faint in Atlantic wolffish liver, which may indicate that the relative proportion of this transcript in this fixed amount of mRNA (1 μg) was reduced, owing to the high concentration of the AFP transcript [59].…”