Developing of isogenic cell lineages with different aleles XPG through transduction with lentiviral vectors

Abstract

DNA is the center of all cells' genetic information and it is constantly exposed to endogenous or exogenous genotoxic agents that can cause damage which eventually lead to mutagenesis or cell death. The most common endogenous agents are reactive oxygen species (ROS), originated mainly in processes such as cellular respiration and inflammation. These agents oxidize bases, sugars, phosphates and causing single strand breaks (SSBs) or double (DSBs) in the DNA molecule. During the evolution, cells have developed DNA repair systems that correct the damage. Dysfunctions in these systems lead to the accumulation of DNA damage and human syndromes are important for understanding processes such as aging and neurodegeneration, linked mainly to the accumulation of damage in both the nuclear and mitochondrial DNA. Several knowledge gaps exist, lacking models to better investigate the causes for these processes. The main goal for this project is to develop isogenic lineages fromXPCS1LV cell line, deleted for the gene encoding for the XPG protein, which has a role as endonuclease in the nucleotide excision repair (NER). Xeroderma pigment sum patients carrying homozygous mutations in the XPG present high skin cancer incidence, as well as clinical phenotypes may also include neurodegeneration and premature aging. This cell lines will be transduced with lent viral vectors carrying the wild-type XPG gene or alleles of XPG mutated genes identified in patients from Cabo Frio, Rio de Janeiro, Brazil and characterized by Dr. Daniela Soltys. Thus, it is expected to obtain four new cell lines, one of these being wild-type, two sensitive only to ultraviolet light while the third one will be sensitive to oxidative stress and UV (transduced with a control vector). Thus we expect to obtain more stable and reliable cell models to identify the XPG relationship to repair the damage caused by oxidative stress and if such damage is related with mitochondrial dysfunction.