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Disease outbreak is harmful to aquaculture
and trade; especially harmful to the salmon
Fish are cultured at high densities and they
are more susceptible to pathogens.
Antibiotics and vaccination have partially
controlled these outbreaks, but there are
concerns of antibiotic resistance.
Another possible approach to improve
disease resistance is enhancing the
immune system of fish by creating
transgenic fish with genes responsible for
immunity in fish.
Lysozyme- antimicrobial/antibacterial
protein that defends fish against
Promotes phagocytosis of bacteria.
Found in mucus, blood, and many other
tissues, especially the kidneys.
Also found in fertilized eggs; may play a
role in preventing transmission of
pathogens to progeny.
Produce transgenic Atlantic salmon that
express lysozyme transgene in multiple
tissues and increase the bacterial lytic
activity of kidney tissue.
Stripped eggs and sperm from collected
Atlantic salmon (Salmo salar).
Cultured fertilized eggs in Heath tray
incubators at 6-8 ºC until the yolk sac
stage and transferred to fresh water
Lysozyme II cDNA isolated from a
rainbow trout (Oncorhynchus mykiss)
liver cDNA library.
Ligated into a gene cassette, resulting in
an ocean pout antifreeze protein
promoter-trout lysozyme fusion gene
construct (opAFP-rtLys)
2186 bp BamHI-HpaI promoter and 5’
untranslated region from the op5aAFP
gene linked to the rainbow trout
lysozyme cDNA
The 3’-end of the construct was ligated
to a 1163 bp fragment from the op5a
AFP gene that included the
polyadenylation and transcription
termination signals
› opAFP promoter
› 5’
Lysozyme gene
Sequenced Lysozyme cDNA and
compared to a sequence found in
Used several different PCR primers and
used locations of PCR amplicons to
determine the sequence of the
Fertilized eggs were microinjected
through the micropyle with 1x10^6
copies of the opAFP-rtLys gene
Transgenic fish were crossed with wild
Atlantic salmon to develop subsequent
Phenotype of transgene expression not
as obvious as a growth hormone
 Screened for lysozyme transgene when
founder generation (P0) reached sexual
 Followed inheritance for three
generations using PCR primers 1848-F
and 244-R.
PCR screening of 100 yolk sac fry from P0
female showed two carried lysozyme
 F1 generation male cross bred with wild
female, PCR of blood samples showed
53% inheritance frequency.
 Next generation showed about 50%
inheritance frequency.
Northern blot analysis revealed mRNA in
gill, kidney and spleen tissues.
 No evidence from non-transgenic
 RT-PCR showed presence of lysozyme
transgene mRNA in all tissues.
A turbidimetric assay for lysozyme
activity was performed using kidney
The lysozyme activity in transgenic
kidney tissue was 40% greater than nontransgenic tissue.
There is no direct evidence that enhanced
lysozyme levels improve the disease
resistance of fish yet.
 Due to a lack of quarantine facilities,
disease challenge tests could not be
performed on the salmon.
 But the evidence for lysozyme transgene
expression and increased levels of kidney
lysozyme activity suggests that the immune
system of transgenic salmon would be
enhanced compared to non-transgenic
Fletcher, G. L., Hobbs, R. S., Evans, R. P.,
Shears, M. A., Hahn, A. L., & Hew, C. L.
(2011) Lysozyme transgenic Atlantic
salmon (Salmo salar L.). Aquaculture
Research, 42: 427-440.