Lysozyme transgenic Atlantic salmon (Salmo salar L.)

Jenna KausnerApril 28, 2011

PBIO 427

Background 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.

Background 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.

Lysozyme 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.

Purpose

Produce transgenic Atlantic salmon that express lysozyme transgene in multiple tissues and increase the bacterial lytic activity of kidney tissue.

Egg collection

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.

Plasmid construction 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)

Gene construct 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

Gene construct

› opAFP promoter Lysozyme gene polyA

› 5’ 3’

opAFP-rtLys

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.

Microinjection

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.

Identification of transgenic salmon

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.

Transgene inheritance

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.

PCR results

Inheritance results

Lysozyme transgene expression

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.

Lysozyme activity

A turbidimetric assay for lysozyme activity was performed using kidney tissue.

The lysozyme activity in transgenic kidney tissue was 40% greater than non-transgenic tissue.

Gamma distribution

Conclusion 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.

Reference

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.