Immortalisation of primary cell lines as tools for investigation into feline diseases
2023
Adamson, Louise | Bergkvist, Gura | Burdon, Tom | Schoenebeck, Jeffrey | EveryCat Health Foundation
Feline tooth resorption (TR) is a disease that is estimated to affect around 60% of domestic cats. This disease is caused by dysregulated odontoclasts, and results in painful, permanent tooth loss in adult cats. There is no current treatment, and research into the disease is limited by the availability of odontoclasts for experimental use. Feline induced pluripotent stem cells (fiPSCs) could provide a limitless supply of multiple cell types including osteoclasts, but to date, routine establishment of fiPSCs has not been achieved. Here we investigate the use of SV40 large T antigen (SV40Tag) as a tool to induce cell immortalization and aid reprogramming to fiPSCs. Feline fibroblasts were extracted from tissue removed during routine castrate procedures. These cells have a finite lifespan, so we transfected them with a doxycycline inducible SV40Tag piggyBac vector. Transfected cell lines had an increase in growth rate and passage numbers beyond their previously observed lifespan. Following optimisation of the transfection procedures, these stable integration lines were used in reprogramming experiments. SV40Tag mouse embryonic fibroblast (MEF) lines were generated as a positive control for reprogramming. Based on colony counts and histology, addition of SV40Tag increases efficiency of Yamanaka factor-based reprogramming in MEF cells. These observations suggest that the addition of SV40Tag has the potential to improve feline somatic cell reprogramming.
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Эту запись предоставил University of Edinburgh