A lacZ-based vector system for the rapid detection of V(D)J recombinase activity
| Autor: | Bernd Bühler, Georges Köhler, Peter J. Nielsen |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
Recombination
Genetic Mice Inbred BALB C Recombinase activity Immunology Genetic Vectors lac operon Mice Transgenic Transfection Biology Molecular biology Blotting Southern Mice Lac Operon Transcription (biology) Cell culture DNA Nucleotidyltransferases Immunology and Allergy Recombination signal sequences Animals Gene VDJ Recombinases Recombination Cells Cultured |
| Zdroj: | Journal of immunological methods. 175(2) |
| ISSN: | 0022-1759 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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