|dc.description.abstract||A neuron is a fundamental unit of the nervous system. These cells are differentiated and are not readily replaced. They are extremely active and display high metabolic rates. Because of their high activity they are subjected to a large amount of DNA damage throughout their lives. The most genotoxic type of DNA damage is a break in both strands of DNA and there can be as many as 10 to 50 double-stranded breaks (DSBs) in every cell per day. Because of this, a high-fidelity DNA repair mechanism would seem critical for ensuring neuronal longevity.
There are two main mechanisms in which DNA DSBs are repaired: homologous recombination repair (HRR) and non-homologous end joining (NHEJ). HRR is said to occur in proliferating cells, whereas NHEJ is said to occur in post-mitotic cells. HRR is a high-fidelity process that uses a homologous copy of damaged DNA, usually from a sister chromatid, as a template to achieve error-free repair. In contrast, NHEJ is an error-prone system that simply uses a resection/re-ligation method to join the two ends of DNA back together. Because neurons are post-mitotic, they are thought to be repaired by NHEJ. However, given their longevity and high levels of activity, it would seem essential that neurons repair DNA DSBs by an error-free system.
The goal of this study was to determine whether HRR occurs in post-mitotic neuronal cells. The cell line Neuro-2a (N2a) is a mouse derived neuroblastoma cell line. The first objective of this project was to determine a differentiation protocol for this cell line. It was found, through cell counting and EdU incorporation assays, that cells treated with media containing 1% foetal bovine serum (FBS) and 10 μM retinoic acid (RA), over the course of seven days, showed the greatest number of N2a cells entering a non-proliferative state.
The plasmids DR-GFP and I-SceI were amplified and purified by bacterial transformation and miniprep. Once the plasmids were purified, DR-GFP was transfected into N2a cells by lipofection. The DR-GFP plasmid is a vector that has been engineered to contain a non-functional full-length green fluorescent protein (GFP) gene with a 5’-premature stop codon/I-SceI recognition sequence. These transfected cells were then treated with puromycin, as the DR-GFP plasmid contains a puromycin resistance gene, to generate a stable N2a-DRGFP cell line.
Once stable integration of DR-GFP was achieved, the N2a-DRGFP cells were then transfected with the I-SceI plasmid. I-SceI restriction endonuclease encoded by the I-Scel plasmid, has no recognition sequence in the mouse genome, so the expression of I-SceI will induce a single DSB at the DR-GFP 5’-premature stop codon/I-SceI recognition site. The DR-GFP vector contains a portion of the wild-type GFP sequence, so if HRR occurs after I-SceI expression, the GFP 5’- premature stop codon will be removed and a functional GFP protein will be detected by immunofluorescence.
No fluorescence was observed after I-SceI transfection in proliferating cells. PCR amplification of a DR-GFP sequence from N2a-DRGFP cells showed that DR-GFP had not been stably integrated into N2a cells. Similarly, co-transfection of I-SceI and DR-GFP into N2a cells also did not show any fluorescence. To determine possible causes for this result, plasmids were sequenced, and it was found that the DR-GFP sequence was incorrect. Because of this, no further experimentation of HRR in post-mitotic cells could occur until the correct DR-GFP plasmid is obtained.||