Sensitive analysis of recombination activity using integrated cell surface reporter substrates.
BACKGROUND
Recombination processes play a crucial role in the functioning of the immune system and are also involved in mutation events that result in various malignancies. So far the study of recombination activity has frequently relied on the use of reporter substrates that are limited by low sensitivity as well as tedious and distorting readout procedures.


METHODS
Immunoglobulin class switch recombination substrates were generated which, upon recombination, resulted in the surface expression of human CD4 or murine MHC class I H-2K(k) and thus allowed for cytometric evaluation.


RESULTS
Recombining cells harboring integrated reporter substrates were analyzed by immunofluorescence and flow cytometry and could easily be isolated by high-gradient magnetic cell sorting (MACS). The analysis was not influenced by cloning efficiencies, as would be the case after drug selection, or prokaryotic recombination that might occur after analysis of recovered substrates 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 recombination 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 of very rare recombination events and thus permits the study of cell types with extremely low recombination activities.
Christine
R
R
Deutsches Rheumaforschungszentrum, Berlin, Germany.
Siebenkotten
G
G
Radbruch
A
A
D000818:Animals; D015704:CD4 Antigens / Q000032:analysis* / Q000235:genetics; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry*; D002460:Cell Line; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D017930:Genes, Reporter; D006183:H-2 Antigens / Q000032:analysis* / Q000235:genetics; D006801:Humans; D017578:Immunoglobulin Class Switching; D018189:Immunomagnetic Separation; D051379:Mice; D011956:Receptors, Cell Surface / Q000032:analysis*; D011995:Recombination, Genetic; D013379:Substrate Specificity
D015704:CD4 Antigens; D006183:H-2 Antigens; C024755:H-2K(K) antigen; D011956:Receptors, Cell Surface