Applications of fluorescence for detecting rare sequence rearrangements in vivo.
Homologous recombination (HR) is an important pathway for the accurate repair of potentially cytotoxic or mutagenic double strand breaks (DSBs), as well as double strand ends that arise due to replication fork breakdown. Thus, measuring HR events can provide information on conditions that induce DSB formation and replicative stress. To study HR events in vivo, we previously developed Fluorescent Yellow Direct Repeat (FYDR) mice in which a recombination event at an integrated transgene yields a fluorescent signal. Recently, we published an application of these mice demonstrating that fluorescent recombinant cells can be directly detected within intact pancreatic tissue. Here, we show that in situ imaging is a more sensitive method for detecting exposure-induced recombinant cells, yielding statistical significance with smaller cohorts. In addition, we show inter-mouse and gender-dependent variation in transgene expression, examine its impact on data interpretation, and discuss solutions to overcoming the effects of such variation. Finally, we also present data on enhanced yellow fluorescent protein (EYFP) expression, showing that several tissues, in addition to the pancreas, may be amenable for in situ detection of recombinant cells in the FYDR mice. The FYDR mice provide a unique tool for identifying genetic conditions and environmental exposures that induce genotoxic stress in a variety of tissues.
Wiktor-Brown
Dominika M
DM
Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachsuttes 02139, USA.
Hendricks
Carrie A
CA
Olipitz
Werner
W
Rogers
Arlin B
AB
Engelward
Bevin P
BP
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D016923:Cell Death / Q000187:drug effects; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005453:Fluorescence; D005786:Gene Expression Regulation; D016678:Genome / Q000235:genetics; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016685:Mitomycin / Q000494:pharmacology; D009928:Organ Specificity; D010179:Pancreas / Q000166:cytology / Q000187:drug effects; D011995:Recombination, Genetic / Q000187:drug effects / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D019076:Transgenes
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D016685:Mitomycin