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Transcript

Disease outbreak is harmful to aquaculture
and trade; especially harmful to the salmon
industry.

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
infection.

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
aquaria.

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
polyA
3’

Sequenced Lysozyme cDNA and
compared to a sequence found in
literature.

Used several different PCR primers and
used locations of PCR amplicons to
determine the sequence of the
integrant.

Fertilized eggs were microinjected
through the micropyle with 1x10^6
copies of the opAFP-rtLys gene
construct.

Transgenic fish were crossed with wild
Atlantic salmon to develop subsequent
generations.
Phenotype of transgene expression not
as obvious as a growth hormone
transgene.
 Screened for lysozyme transgene when
founder generation (P0) reached sexual
maturity.
 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
transgene.
 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
controls.
 RT-PCR showed presence of lysozyme
transgene mRNA in all tissues.


A turbidimetric assay for lysozyme
activity was performed using kidney
tissue.

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
salmon.


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.