The Effects of Transfected Mutant and Wild Type Rag Genes in Different Cell Types

Abstract

The ability to perform V(D)J recombination can be transferred to non-lymphoid cells by stable or transient transfection of the RAG genes. This fact was used to test the effect of wild type and mutant RAG-genes on the V(D)J recombination activity in recombination competent and incompetent cells. Cotransfection of wild type RAG genes induced V(D)J recombination in NIH 3T3 fibroblasts, but had no effect in the pre B cell line 38B9 and even reduced the recombination activity of the fibroblast cell line L4 which has been stably transfected with both RAG genes, indicating that these cell lines lack co-factors which are necessary for higher recombination activity. A mutant RAG-1 gene which lacks a part of the topoisomerase I-like region and a mutant RAG-2 gene which lacks its 89 C-terminal amino acids did not activate V(D)J recombination in NIH 3T3 cells. They did not decrease the recombination activity in L4 cells, but increased the V(D)J recombination activity in 38B9 cells, probably by protecting endogenous wild type RAG mRNAs and proteins against RNAses and proteinases. Treatment respectively co-transfection of NIH 3T3 cells with the V(D)J recombination stimulating agent caffeine and the lymphoid specific transcription factor Oct2A did not lead to induction of V(D)J recombination in the absence of RAG genes.