Browsing by Author "Escorza Sius, Amanda Paz"

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    Efficient transfection protocol of rainbow trout gills epithelial (RTgill-W1) cell line through nucleofection system. v1
    (2024) Escobar Aguirre, Sebastián Gonzalo; Escorza Sius, Amanda Paz; Escobar Aguirre, Matías
    In this protocol, we optimize the experimental condition to express plasmid DNA in salmonid fish rainbow trout gills epithelial (RTgill-W1), using a nucleofection system. The principle of nucleofection is a combination of electrical parameters that ensures efficient DNA delivery into the nucleus, combined with low toxicity and high cell viability. Using the Neon Transfection System (Invitrogen‱ MPK5000; code: 10431915) and the Invitrogen‱ Neon‱ Transfection System Kit 10 μl (brand: Invitrogen‱ MPK1096; code: 10124334) we were able to express over 60% of GFP in RTgill W 1 line cells. These results were achieved after trying different optimization steps voltage (ranges between 850 - 1600 volts), pulse exposure (times 10 30 ms), and number of pulses, being the condition 1600 volts, 20 ms 1 pulse, and 1 μg μL of plasmid the most effective compared to the other conditions, presenting a higher percentage of GFP expression and lower mortality rate.
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    Transferrin knockout reveals a tolerance phenotype against Piscirickettsia salmonis in Atlantic salmon phagocytes
    (2025) Mejías, Madelaine; Sáez Beltrán, Alejandro José; Escorza Sius, Amanda Paz; Galdames Contreras, Felipe; Jin, Yehwa; Dettleff Faundes, Phillip Jame; Ojeda, Ingrid; Pulgar, Rodrigo; Escobar Aguirre, Sebastián
    Salmonid rickettsial septicemia (SRS), caused by Piscirickettsia salmonis, is a major challenge in Chilean aquaculture. We evaluated the impact of the vaccine on fish survival, bacterial load, and iron metabolism-related gene expression under field conditions. Atlantic salmon received either a pentavalent inactivated vaccine plus a live attenuated P. salmonis vaccine (SIA) or a tetravalent control vaccine (SS). While survival was similar early (≤ 28 weeks post-seawater transfer), SIA-vaccinated fish showed greater survival by week 41 (85% vs. 52%). Quantitative PCR confirmed a reduced bacterial load in the SIA group during active infection. Expression analysis revealed distinct temporal regulation of iron metabolism genes: ferritin and hepcidin were upregulated in freshwater, whereas transferrin and its receptor were elevated in seawater in the SIA group, suggesting a link between iron homeostasis and vaccine-induced protection. To further investigate the role of transferrin, we generated transferrin-knockout (TF-KO) phagocytes using CRISPR/Cas9. In vitro infection assays revealed that, compared with wild-type (TF-WT) cells, TF-KO cells presented reduced cytopathic effects, decreased formation of P. salmonis-containing vacuoles (PCVs), and improved viability. Surprisingly, no differences in bacterial load or iron-related gene expression were detected between TF-KO and TF-WT cells, indicating that transferrin disruption did not directly alter iron homeostasis. Global transcriptomic analysis revealed 311 differentially expressed genes in TF-KO cells, with functional enrichment in metal-binding and zinc-dependent processes but no direct association with iron metabolism. These findings suggest that transferrin deficiency confers an infection-tolerant phenotype through transcriptional reprogramming unrelated to iron regulation, highlighting novel mechanisms of host‒pathogen interactions and potential avenues for SRS control in aquaculture.