Enumeration of double stranded DNA breaks in developing thymocytes in young and aged mice (B1)

Abstract

Abstract Aging at the cellular and molecular level has been shown to involve a decline in the immune system. In spite of this decline, the aged thymus is still functional in both thymocyte development and output of mature naïve T cells producing a small but significant population. During V(D)J recombination double stranded breaks (DSB) are created in the DNA of developing thymocytes. Complete and accurate resolution of the DSB is critical to the proper function and survival of cells. In our studies we utilized gamma phosphorylated-H2A.X(γH2A.X) to estimate the amount of DSB that are created and/or persist in thymocytes. H2A.X phosphorylation is the earliest ATM-dependent response to DSB, and it is speculated that the γH2A.X may help keep broken DNA ends together. For quantitation it is import to note that many H2AX molecules are phosphorylated in response to one DSB. The amplification of the signal allows us to visualize foci. We find cells from aged thymus have more DSB than cells from the young thymus. This increase in breaks may be the result of more breaks being created or breaks persisting due to slow or lack of DSB repair. By utilizing fluorescent staining and confocal microscopy we were able to further investigate the number of cells with DSBs and compare different thymocyte subpopulations. Here we show the enumeration of γH2A.X foci in thymocyte subpopulations in both young and aged mice.