Sensitive analysis of recombination activity using integrated cell surfacereporter substrates

Background: Recombination processes play a crucial role in the functioning of the immune system and are also involved in mutation events that result i n various malignancies. So far the study of recombination activity has freq uently relied on the use of reporter substrates that are limited by low sen sitivity as well as tedious and distorting readout procedures. Methods: Immunoglobulin class switch recombination substrates were generate d which, upon recombination, resulted in the surface expression of human CD 4 or murine MHC class I H-2K(k) and thus allowed for cytometric evaluation. Results: Recombining cells harboring integrated reporter substrates were an alyzed by immunofluorescence and flow cytometry and could easily be isolate d by high-gradient magnetic cell sorting (MACS). The analysis was not influ enced by cloning efficiencies, as would be the case after drug selection, o r prokaryotic recombination that might occur after analysis of recovered su bstrates in bacteria. In addition, cytometric readout is much faster, as it can be performed immediately after recombination. The substrate exhibited properties compatible with the detection of immunoglobulin class switch rec ombination and permitted the detection of recombination events down to 10(- 5) per cell and generation. Conclusions: The high sensitivity of this system allows precise detection o f very rare recombination events and thus permits the study of cell types w ith extremely low recombination activities. Cytometry 37:205-214, 1999. (C) 1999 Wiley-Liss, Inc.