A lacZ-based vector system for the rapid detection of V(D)J recombinase activity.
Episomal vectors have been developed which are useful for studying V(D)J recombination both after transient transfections and in stably transfected cells. In contrast to recombination substrates previously described for transient assays, rearrangement of these vectors results in expression of beta-galactosidase which can be visualized directly in the transfected cell, shortening the time required for the assay to 1-2 days instead of 3-4 days. When these substrates are stably integrated into a preB cell line, subclones are found which show no beta-galactosidase staining, although the substrate is properly integrated, transcriptionally active and the transfectants still possess recombinase activity. This finding suggests that, at least in some chromosomal locations, transcription through a locus bearing recombination signal sequences is not sufficient for V(D)J recombination. Using these same vectors, we estimate that the frequency with which V(D)J recombination-negative preB variants arise is less than 10(-4) per generation.
Bühler
B
B
Max-Planck-Institut für Immunbiologie, Freiburg, Germany.
Köhler
G
G
Nielsen
P J
PJ
D000818:Animals; D015139:Blotting, Southern; D002478:Cells, Cultured; D004254:DNA Nucleotidyltransferases / Q000032:analysis*; D005822:Genetic Vectors; D007763:Lac Operon / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D011995:Recombination, Genetic / Q000276:immunology*; D014162:Transfection; D045567:VDJ Recombinases
D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases