? Applications of """"""""Recombomice"""""""" for Cancer Research ? ? Every time a cell divides, billions of base pairs of information must be accurately copied in the face of an onslaught of DNA damage. Homology directed repair (HDR) provides one of the most important mechanisms for coping with damaged DNA. If coding information is missing or corrupted, HDR can extract sequence information available elsewhere in the genome. Although HDR is generally beneficial, transfer of genetic information is risky, since misalignments can lead to tumorigenic rearrangements. To investigate the process of HDR in vivo, we have engineered the first mouse model in which HDR can be detected in somatic cells by the appearance of a fluorescent signal. In the fluorescent yellow direct repeat (FYDR) recombomice, recombination at an engineered substrate yields fluorescence. Recombination assays are simple and rapid, making it possible to do in days what used to take weeks. In addition, the FYDR mice overcome limitations of previous systems. For example, although APRT mice can be used to detect loss of heterozygosity, technically demanding assays are necessary to identify HDR events; in the pun mice, only embryonic recombination events can be detected. In contrast, FYDR mice yield a fluorescent signal that is specific to HDR events, and the recombination rate can be readily measured in cells from both embryonic and adult tissues. Furthermore, fluorescence makes it possible to capture in situ images of recombined cells, making it possible to discern independent lineages of recombinant cells in vivo.
Our Specific Aims are to I) Evaluate the frequency of recombinant cells in multiple tissues; II) Develop methodology for quantification of recombinant pancreatic cells in situ and reveal the relative frequency of recombinant cells among two different cell types within a normal tissue for the first time; III) Measure the effects of environmental factors on recombination in vivo; and IV) Reveal how specific genes (Blm and p53) affect recombination susceptibility in vivo. The broad long term objectives of this work are to demonstrate the utility of this newly developed technology for studying recombination in mammals, to substantially expand the capabilities of the existing system, and to elucidate environmental and genetic factors that influence a person's susceptibility to spontaneous, environmentally-induced, and cancer therapy-induced DNA rearrangements. ? ? ? ?
