Biomedical subjects

G Taccioli

Publications and source records attributed to G Taccioli.

3 recordsLinked to original sources

PubMed1993

A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination.

Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer; substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.

Animals↗

PubMed1992

Function and control of recombination-activating gene activity.

The RAG-1 and RAG-2 genes synergistically confer VDJ recombinase activity to nonlymphoid cell lines. To unequivocally test RAG gene function, we created lines of mice that lack functional copies of these genes. Consistent with the possibility that RAG gene encode the tissue-specific components of VDJ recombinase, RAG-2-deficient mice are viable but have a severe combined immune deficiency due to inability to initiate VDJ recombination and thereby generate mature lymphocytes. RAG-2-deficient mice have no obvious defect in any tissue or lineage other than lymphocytes, indicating that VDJ recombinase activity and RAG-2-gene function is required only for lymphocyte development. Levels of RAG-1 and RAG-2 expression in primary murine lymphoid tissues and lymphoid bone marrow cultures generally are much higher than those of transformed precursor B-cell lines. Low-level RAG gene expression in permanent cell lines results from a decline during propagation due to outgrowth of cells with lower RAG expression levels. The low and variable level of RAG gene expression in transformed pre-B cell lines correlates with low and variable rates of endogenous VDJ recombination; therefore, such lines are not reliable models for experiments aimed at studying mechanisms that target this activity to particular variable region gene segments. To generate such a system, we introduced RAG genes into B-lineage lines under the control of a heat shock-inducible promoter; heat-shock treatment induces extremely high-level but transient RAG expression accompanied by parallel induction of VDJ recombinase activity. Such cells efficiently rearrange transfected VDJ recombination substrates in a regulated manner that is dependent on the activity of transcriptional control elements associated with the target V gene segments.

Animals↗

PubMed1992

VDJ recombination.

The ability of lymphocyte receptor V, D and J gene segments to rearrange generates much of the receptor diversity that is the hallmark of the immune system. Naturally, the mechanisms of immunoglobulin and T-cell receptor gene recombination are of enormous interest. Here, Fred Alt and colleagues review current understanding of the process and speculate on future findings.

Animals↗