Development and application of a novel in vivo EGFP based V(D)J recombination assay
LicenseStandard (Fassung vom 03.05.2003)
A single cell in vivo green fluorescence assay was successfully developed to monitor effectively V(D)J recombination efficiency. Our results show that the in vivo V(D)J recombination efficiency is essentially low (ca. 1 %) in 293 cells even though the transfection efficiency reached 60 % to 90 %. This in vivo V(D)J recombination efficiency correlated well with that achieved by the classical in vitro V(D)J recombination assay. The in vivo V(D)J recombination efficiency depended on the relative RAG-1 and RAG-2 expression vector concentrations used in co-transfection, but was independent of relative Artemis expression vector concentrations. The novel in vivo EGFP V(D)J recombination assay has been successfully set up and can in the future be applied for testing the efficiency of V(D)J recombination in cell lines, for evaluating roles of components in V(D)J recombination, for searching for novel factors involved in the V(D)J recombination process and for diagnosing SCID patients.