The goal of this proposal is to develop and apply an innovative technique to assess the mitotic history of human T cells on a single- cell basis. The proposed new methodology will exploit the attributes of a novel nucleic acid probe (termed a molecular beacon) in the context of conventional multicolor immunocytometry by FACS to determine telomeric terminal restriction fragment (TRF) length, which is a direct reflection of T cell mitotic history and proliferative potential. To achieve this research goal, the PI plans to: 1) optimize the design of a hairpin molecular beacon to quantitate the hexameric repeats of telomeric TRF; 2) refine the fixation and hybridization protocols for quantitative assessment in PBMC samples in suspension; 3) validate TRF lengths using cells sorted on beacon intensity; 4) compare different beacon conjugates to optimize compatibility with available commercial antibodies for cell surface markers; and 5) correlate FACS quantitation of telomeric repeats with the replicative capacity of T cells in vitro. To demonstrate the research applicability of this new method, ex vivo analysis will be performed on PBMC samples from patients with Recurrent Pregnancy Loss (as a disease model) and from patients with HIV infection.
