CRISPR/Cas9 based knockout of genes in SHK-1 cell line, to investigate their role in development of Infectious salmon anemia virus infection

In recent years, we have realized the potential of aquaculture as an industry to meet the goal of rising global food demand in a cost-effective manner, rooted in sustainable and environment friendly practices. Atlantic salmon, as a fish, has a sizeable share in the Norwegian Aquaculture industry and plays a popular role in the Norwegian food culture. Out of the several diseases that affect the health of Atlantic salmon, the viral disease caused by Infectious Salmon Anemia virus is particularly notorious. It is a notifiable disease as per the World Organization for Animal Health and infection with ISAV has known to be difficult to control, causing epizootic episodes that threaten not only threaten the welfare of commercial salmons but also wild populations. Some of the challenges involved with control of the ISAV infections include a high mortality rate and lack of any treatment options – prophylactic or otherwise, leading to severe economic losses to the Norwegian aquaculture industry. In this study we aimed to identify the genes involved in the development of ISAV infection. We investigated if at least one of the three genes- TMEM199, WDR7 and CCDC115 could be involved in the mechanism of infection for ISA virus. This was achieved by a CRISPR/Cas9 based knockout of the target genes followed by virus challenge study on SHK-1 cell line.M-AB

Outlook Magazine, Fall 2005

https://digitalcommons.wustl.edu/outlook/1161/thumbnail.jp

Evaluation of SpliceAI for Improved Genetic Variant Classification in Inherited Ophthalmic Disease Genes

ABSTRACT EVALUATION OF SPLICEAI FOR IMPROVED GENETIC VARIANT CLASSIFICATION IN INHERITED OPHTHALMIC DISEASE GENES By Melissa Jean Reeves, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2023 Major Director: Melissa Jamerson, PhD, MLS(ASCP) Associate Professor, Department of Medical Laboratory Sciences Inherited ophthalmic diseases impact individuals around the globe. Inherited retinal diseases (IRDs) are the leading cause of blindness in individuals aged 15 to 45. The personal, social, and economic impact of vision loss is profound. Due to individual differences, symptoms can be variable, and it may be difficult to diagnose some diseases based on phenotype alone. Clinicians often seek out genetic testing to confirm clinical diagnoses when other avenues have failed. Clinical laboratories use all available data, such as frequency, population, or computational data, to evaluate genetic variants and determine their classification. Clinical laboratories may not have enough evidence to classify a genetic variant as pathogenic or benign when testing is performed, so variants may be classified of uncertain significance. Because inherited retinal diseases are considered rare, there are limited treatments available, and most treatment is offered through clinical trials. Clinical trials often have stringent inclusion and exclusion criteria to ensure the most optimum outcome for the study. Due to constraints of a study, patients often must have definitive genetic results to qualify for a trial. A variant of uncertain significance would likely disqualify an individual for a clinical trial. Functional assays, such as the minigene assay, have been used extensively across multiple genes and diseases with ease. This study aimed to investigate a novel methodology for the minigene assay and establish the sensitivity of SpliceAI for predicting synonymous splice effects in variants with a SpliceAI change (∆) score ≥ 0.8 in inherited ophthalmic disease genes. This study used the “P” or process component of the Structure-Process-Outcome (SPO) Donabedian model to evaluate the addition of the minigene assay to the clinical testing workflow. This study also highlights the importance of using a well-validated framework, such as Donabedian, in conjunction with clinical laboratory quality improvements. Of the 617 synonymous variants in 20 ophthalmic disease genes targeted in the database, 86 synonymous variants in 14 genes were scored ≥ 0.8. Twenty synonymous variants in two ophthalmic disease genes (ABCA4 and CHD7) were selected for this preliminary study. Twenty wildtype and variant pairs were assessed using the novel minigene test to review splice outcomes. This study established that this novel minigene test could be used in a clinical laboratory as a part of the clinical testing pipeline. Of the 20 variants targeted, 14 variants could be evaluated by minigene. Six variants did not produce high-quality data and will need to be repeated. Eleven of the 14 variants reviewed showed aberrant splice effects through the minigene assay, matching the SpliceAI prediction. Three variants matched the wildtype transcript and were therefore considered discordant. Based on these results, the sensitivity of SpliceAI for predicting splice effects in synonymous variants in inherited ophthalmic diseases is approximately 79%, slightly less than the expected 80%. The shift in sensitivity is likely due to the small sample size in this study. A Fisher’s exact test was performed to evaluate the concordance rate between minigene outcomes and SpliceAI predictions with a p value of 0.2222, indicating no statistical difference between SpliceAI predictions and minigene outcomes. The results of this study indicate that SpliceAI has a predictive efficiency in ophthalmic disease genes of 79%, which is well below what would be needed (\u3e 95%) for a clinical laboratory to rely solely for variant classification. Though the predictive efficiency is less than expected, this preliminary study offers insight into the predictive value of SpliceAI for synonymous variants in inherited ophthalmic disease genes. This study also introduces a novel minigene method that other clinical laboratories across other diseases and genes can reliably use

2011-2012 annual report to the S.C. General Assembly and the S.C. Budget & Control Board

The South Carolina Centers of Economic Excellence publishes an annual report to the South Carolina Budget & Control Board with description of the SmartState program, snapshot of company partners, description for each Centers of Economic Excellence, program achievements, review board members, and COEE Council of Chairs members

Activities of the Finnish Institute of Occupational Health in 2009–2013 : Self-evaluation

This self-evaluation report describes FIOH’s activities in 2009–2013. It has been produced for the international evaluation of FIOH, which was carried out in 2014 by an independent international evaluation group of experts, appointed by the Ministry of Social Affairs and Health of Finland

Stories in Agriculture and Life Sciences, Spring 2017

Stories we hope will inform and inspire you. Stories about how the college is constantly striving to make new discoveries, encourage and educate students and serve Iowans. Stories of the people who make this college one of the premier institutions of agriculture and life sciences in the world

Tracking and analysis of movement at different scales: from endosomes to humans

Movement is apparent across all spatio-temporal scales in biology and can have a significant effect on the survival of the individual. For this reason, it has been the object of study in a wide range of research fields, i.e. in molecular biology, pharmaceutics, medical research but also in behavioural biology and ecology. The aim of the thesis was to provide methodologies and insight on the movement patterns seen at different spatio-temporal scales in biology; the intra-cellular, the cellular and the organism level. At the intra-cellular level, current thesis studied the compartmental inheritance in Human Osteosarcoma (U2-OS) cells. The inheritance pattern of the endosomal quantum dot fluorescence across two consecutive generations was for first time empirically revealed. In addition, a in silico model was developed to predict the inheritance across multiple generations. At the cellular level, a semi-automated routine was developed that can realize long-term nuclei tracking in U2-OS cell populations labeled with a cell cycle marker in their cytoplasm. A method to extract cell cycle information without the need to explicitly segment the cells was proposed. The movement behaviour of the cellular population and their possible inter-individual differences was also studied. Lastly, at the organism level, the focus of the thesis was to study the emergence of coordination in unfamiliar free-swimming stickleback fish shoals. It was demonstrated that there exist two different phases, the uncoordinated and the coordinated. In addition, the significance of uncoordinated phase to the establishment of the group’s social network was for first time evinced. The adaptation of the stickleback collectives was also studied over time, i.e. the effect of group’s repeated interactions on the emergence of coordination. Findings at the intra-cellular and cellular level can have significant implications on medical and pharmaceutical research. Findings at the organism level can also contribute to the understanding of how social interactions are formed and maintained in animal collectives