Prof. Eric Blair, Biology, Leeds
Recombinant Adenoviruses (rAds) are frequently-used vectors for gene therapy and as such are often grown in p53-deficient cells. PTEN is a tumour suppressor gene that encodes a lipid and protein phosphatase enzyme. Loss of PTEN function by mutation increases cell proliferation. It is not known if PTEN has a critical involvement in viral replication and if PTEN loss might be a target for a new generation of rAds. This project is based on a successful final year project where CRISPR-Cas9 technology was used to create a population of epithelial A549 cells in which PTEN protein expression was reduced by >80%. This project will involve now, single cell cloning of the A549/PTEN cells and validation of PTEN expression by PCR and western blot, viral plaque assays to determine whether rAd replicates more efficiently, and monitoring of rAd-specific proteins using immunofluorescence and western blotting of Ad capsid proteins and non-structural proteins (e.g. E1A). Students will be expected to present their findings orally at a research day in York in September 2024.