A lymphoid-specific template independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT) is thought to participate in creation of diversity in the immune system. The expression of TdT is restricted to pre-B and pre-T cells and their differentiation-arrested leukemic counterparts. The mechanisms governing its tissue-specific expression and its unique temporal expression during development are not known. In addition, regulation of expression of the gene in leukemic cells is not understood. Moreover its precise role in generation of the immune repertoire remains to be elucidated. We have constructed full-length human cDNA probes and isolated the entire TdT gene including putative regulatory sequences. TdT DNA constructs have been prepared for cell transfection and transgenic animal studies. He have proposed an experimental strategy to define regulation of TdT gene expression: DNA sequences and protein factors that modulate transcription in normal and leukemic cells will be described and characterized. Regulatory DNA sequences identified in these studies will be introduced into the mouse genome via the construction of transgenic animals in order to characterize their in Vivo function. Transgenic animals carrying control chimeric reporter genes. have been produced and demonstrated to harbor the transgene. The precise roles of TdT in the immune system will be probed through the construction of TdT-deficient mice. TdT genes containing deletions within the coding region will be introduced into mouse genomes via homologous recombination. The resulting heterozygous and homozygous TdT deficient mice will then be screened for gross morphological alterations in lymphoid organs, alterations in the spectrum of T and B cell subsets, and variations in the immunoglobulin repertoire. The precise role of TdT in adding n regions at VDJ junctions will be determined. The long range objective of this proposal is to generate information that will ultimately lead to a comprehensive model for TdT gene expression and function.
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