FT2KO1-L
Salmo salar
Protocols: Tissue samples were immediately frozen at in liquid nitrogen and stored at -80 until further processing Two diets were used in the feeding trial, a standard commercial diet (Nutra Olympic, Skretting Nutreco Company) with relatively high levels of EPA and DHA (see publication), and a low-PUFA diet (SPAROS, LDA.) with reduced levels of DHA and EPA but high composition of 18:2n-6 and 18:3n-3. The feeding trial was carried out at the Institute of Marine Research Matre, Norway, from late January to mid-May 2017, in fresh water at 8-10C with oxygen saturation of 95.3% to 97.7% at outlet during the trial. All experimental fish were initially reared on the commercial diet to Atlantic salmon parrs of approximate weight of 20 g before feeding trial. Groups of 15 elovl2 KOs and 15 wildtypes (WTs) were randomly allocated to 3 tanks and fed low-PUFA diet. The 4th tank containing 30 elovl2 KOs and 30 WTs was fed standard commercial diet. The feeding was done by automatic feeders (Arvotec single feeder). Tissues were sampled after 60 (intermediate) and 108 days (final) of feeding. Fish were anesthetized with Finquel MS-222 (ScanVacc) and then killed with a blow to the head. CRISPR/Cas-9 knock out: Using genomic and cDNA sequences for elovl2 (NC_027318.1, NM_001136553.1), three target sites (T1-3) were selected using a custom made Perl script, which identifies unique CRISPR-target sites. Candidate target sequences were screened against the latest salmon genome assembly (GCA_000233375.4) to eliminate off-target lesions. Salmo salar eggs were injected with a mixture of 50 ng/μl gRNA each for slc45a2 (marker) and elovl2, and 150 ng/μl Cas9 mRNA in Hepes buffer using the picospritzer III (Parker Automation, UK). Microinjected embryos were incubated at 6-8C until hatching. A few weeks after start feeding, fully albino fish were sorted out, fin clipped or fully sampled to check for mutations in elovl2. Salmon were sampled at developmental stage: parr (approxmiately 6 months to 3 years of age) and of unrecorded gender. RNA was extracted using the RNeasy Plus Universal Tissue Kit (Quiagen) Stranded RNAseq libraries were prepared from 1µg total RNA from liver tissue using TruSeq Stranded mRNA library preparation kit (Illumina, San Diego, USA) using double unique indices (#20022371), according to the manufacturer's instruction (Part 15031057 Rev.E).
ENA-FIRST-PUBLIC
2019-04-30T04:02:44Z
ENA-LAST-UPDATE
2018-09-20T14:22:23Z
External Id
SAMEA4932620
INSDC center name
Centre for Integrative Genetics (CIGENE), Norwegian University of Life Sciences, As NO-1432, Norway
INSDC first public
2019-04-30T04:02:44Z
INSDC last update
2018-09-20T14:22:23Z
INSDC status
public
Submitter Id
E-MTAB-7220:FT2KO1-L
broker name
ArrayExpress
common name
Atlantic salmon
developmental stage
parr
diet
low PUFA
genotype
elovl2/slc45a2 CRISPR/Cas9 mediated double knock out
growth condition
fresh water tank
organism part
liver
sample name
E-MTAB-7220:FT2KO1-L
scientific_name
Salmo salar