Development and Characterization of v-myc/v-raf-Transformed Murine Fetal Thymocyte Cell Lines

Transformed murine fetal thymocyte cell lines were derived by incubating fetal thymic organ cultures with a v-myc/v-raf-containing retroviral construct in order to model developmental stages within the early triple negative (CD3−CD4−CD8−) thymocyte population. The resulting 10 cell lines had a lymphoid morphology, were all CD44+, CD90+, and were triple negative by surface antigen analysis. The cell lines, however, were distinguishable by differences in the expression of T cell-associated and T cell-specific genes. The CD3 genes were observed to be discoordinately expressed, in that CD3γ chain gene expression was detected in 2 cell lines in the absence of CD3δ and ϵ expression. Expression of the CD3γ chain gene was observed in cell lines without the expression of other T cell-specific genes or T cell receptor rearrangement and may be one of the earliest T cell-specific genes to be expressed. The transcription factor Ikaros was expressed in all 10 cell lines, whereas the transcription factor TCF1α was expressed only in the 2 most differentiated lines. In 8 cell lines, expression of partial TCR β and/or TCRα transcripts was observed by Northern blot. In several lines, expression of rearranged TCRα transcripts in the absence of TCRβ transcripts was demonstrated; however, TCRβ DJ rearrangements were observed by Southern blot in all but 1 of these cell lines. Thus, these cell lines, ordered based on the general pattern of additive gene expression observed, may reflect various stages of triple-negative thymocyte differentiation and provide anin vitromechanism to elucidate some of the molecular events involved in early thymocyte development.