23 research outputs found
Enhanced transfection of cell lines from Atlantic salmon through nucoleofection and antibiotic selection
Background
Cell lines from Atlantic salmon kidney have made it possible to culture and study infectious salmon anemia virus (ISAV), an aquatic orthomyxovirus affecting farmed Atlantic salmon. However, transfection of these cells using calcium phosphate precipitation or lipid-based reagents shows very low transfection efficiency. The Amaxa Nucleofector technology™ is an electroporation technique that has been shown to be efficient for gene transfer into primary cells and hard to transfect cell lines.
Findings
Here we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry. Depending on the plasmid promoter, TO cells could be transfected transiently with an efficiency ranging from 11.6% to 90.8% with good viability, using Amaxa's cell line nucleofector solution T and program T-20. A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells.
Conclusions
The results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin
The Crystal Structure and RNA-Binding of an Orthomyxovirus Nucleoprotein
Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza
viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as
ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them
to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious
salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular
structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution,
we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic
oligos. Our RNA binding analysis revealed that each ISAV-NP binds ,12 nts of RNA, shorter than the 24ヨ28 nts originally
estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by
results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have
similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and
selective RNP packaging may be accomplished through direct RNA-RNA interactions
TargetFish - Targeted disease prophylaxis in European fish farming
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