A novel fluorescence-based system for assaying and separating live cells according to VDJ recombinase activity.
We describe two retroviral vector-based recombination substrate systems designed to assay for lymphoid VDJ recombinase activity in cultured cells. Both substrates incorporate a constitutive dominant marker gene (the simian virus promoter-driven neo gene) to allow selection of cells that stably integrate the substrate. Both substrates also include a second marker gene that becomes transcriptionally active only when inverted by a site-specific recombination event between flanking immunoglobulin variable-region gene segments. The first vector, similar in structure to previous retrovirus-based recombination substrates, utilizes the bacterial guanine-xanthine phosphoribosyltransferase gene (gpt) as its activatable marker; detection of inversion (VDJ recombinase activity) involves drug selection and Southern blotting analyses. We have used this vector to make a more extensive and quantitative survey of VDJ recombinase activity in B-lineage cell lines than has previously been performed with stable substrates, and we have compared our results with those of other studies that use transient recombination substrates. In the second vector, the activatable gene is the bacterial beta-galactosidase gene (lacZ). Detection for inversional activation of this gene is achieved by a fluorogenic assay, termed FACS-Gal, that detects beta-galactosidase activity in viable cells. The latter assay has the unique advantage of rapidly detecting cells that undergo recombination and also allows viable sorting of cells on the basis of the presence or absence of VDJ recombinase activity. We have used the lacZ vector to rapidly quantitate VDJ recombinase activity in B-lineage cell lines and compared the results with those obtained with the gpt vector. We have also used the lacZ vector to isolate variant pre-B-cell lines with low and high levels of VDJ recombinase activity.
Yancopoulos
G D
GD
Howard Hughes Medical Institute, New York, New York.
Nolan
G P
GP
Pollock
R
R
Prockop
S
S
Li
S C
SC
Herzenberg
L A
LA
Alt
F W
FW
D000818:Animals; D000916:Antibody Diversity; D001402:B-Lymphocytes / Q000276:immunology; D002460:Cell Line; D002999:Clone Cells; D004254:DNA Nucleotidyltransferases / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin / Q000235:genetics*; D007133:Immunoglobulin Joining Region / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008214:Lymphocytes / Q000276:immunology*; D045567:VDJ Recombinases
D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases