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A new approach to understanding T cell development: the isolation and characterization of immature CD4-, CD8-, CD3- T cell cDNAs by subtraction cloning

Orr, Sally L. and Gese, Elaine and Hood, Leroy (1992) A new approach to understanding T cell development: the isolation and characterization of immature CD4-, CD8-, CD3- T cell cDNAs by subtraction cloning. Molecular Biology of the Cell, 3 (7). pp. 761-773. ISSN 1059-1524.

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During T cell development in the mammalian thymus, immature T cells are observed that lack the cell surface markers CD4, CD8, and CD3. A subtracted cDNA library was constructed to isolate cDNAs that are specific for these immature T cells. Tissue-specific expression of 97 individual cDNAs were examined using different cell types by Northern blot analysis, and six cDNAs were analyzed by reverse transcriptase (RT) polymerase chain reaction (PCR) detection of RNA. Approximately 50% of the clones could not be detected on Northern blots, and 40% of the clones were expressed by at least one other cell-type including monocytes, mature T cells, and B cells. Eight cDNA clones appear to be specific for the CD4-, CD8-, CD3- T cell line, used to construct the library, as determined by Northern blot analysis. In addition, 330 cDNA clones were subjected to partial automated DNA sequence determination. Database searches, with both nucleotide and protein translations, revealed cDNAs that exhibit interesting similarities to human cell-cycle gene 1, platelet-derived growth factor receptor, c-fms oncogene (CSF-1) receptor, and members of the immunoglobulin gene superfamily. This approach of employing subtraction coupled with large scale partial cDNA sequence determination can be useful to identify genes that may be involved in early T cell growth, cellular recognition or differentiation.

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Additional Information:Copyright © 1992 by The American Society for Cell Biology Submitted March 26, 1992; Accepted May 28, 1992 We gratefully acknowledge Linda Sakimura and Julie Bars for expert technical help. We thank Dr. Joan Kobori and Dr. Carmie Puckett for critical reading of the manuscript, and Dr. Ellen Rothenberg for the kind gift of the IM7.8.1 and M1.69 antibodies. Also Dr. Ellen Rothenberg and Dr. Jim Allison for the gift of the 705 cell line. We thank Rochelle Diamond and Pat Koen for expert technical assistance for the cytofluorographic analysis and members of the Caltech Division of Biology Sequence Analysis Facility for assistance with the VAX computer and the GCG software. This work was supported by National Institutes of Health grant HD-07257 and the Lucille P. Markey Charitable Trust (S. Orr).
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Deposited On:18 Jul 2006
Last Modified:26 Dec 2012 08:56

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