Hostert, Arnd Martin; (1998) Identification of regulatory elements in the CD8 gene locus. Doctoral thesis (Ph.D), University College London.

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Abstract

The murine CD8 molecule is a disulphide linked heterodimer expressed on thymocytes and some T cells. It is composed of a CD8α and a CD8β chain, both members of the immunoglobulin gene superfamily, with a total molecular mass of 70 kilodalton. Its expression is developmentally regulated and plays an important role during thymocyte development and cytotoxic lymphocyte effector function. The murine CD8 gene locus consists of the CD8α gene downstream of the CD8β gene separated by 36 kilobases with the two genes organized in the same transcriptional orientation. The CD8 gene locus was cloned from genomic cosmid and P1 libraries. A 80kb P1 clone, ICRFP703B21317 (P1-5), was isolated that contains the CD8 genes and flanking DNA sequences. DNasel hypersensitivity analysis carried out using DNA isolated from DNasel treated thymocyte nuclei detected four clusters of DNasel hypersensitive sites (DNasel-HSS) in the CD8 gene locus (CI - CIV). Analogous analysis using DNA isolated from DNasel treated liver nuclei showed three of the four clusters to be thymocyte specific (CII - CIV). Transgenic mice carrying the cloned CD8αβ genomic locus and containing the identified DNasel-HSS clusters express the transgenic CD8α in a developmentally regulated, tissue specific and CD8 T cell subset specific manner. Northern blot analysis of CD8β RNA showed that the transgenic CD8β gene was also expressed only in those tissues which were positive for the endogenous CD8β RNA. Deletion analysis of the identified DNasel-HSS clusters has been carried out using transgenic mice. Fluorescent activated cell sorting analysis of thymocytes from transgenic mice carrying two clusters of DNasel-HSS located 25kb downstream and immediately upstream of the CD8α gene (CI and CII respectively) do not express the transgenic CD8α allele. In contrast, transgenic mice generated with DNasel-HSS cluster CIII, located in the intergenic region, linked to either a murine CD8α or human CD2 reporter gene express the transgenic reporter in a subset specific manner in mature CD8 single positive cells.

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