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|Title:||Use of the 5'-flanking region of the mouse perforin gene to express human Fc gamma receptor I in cytotoxic T lymphocytes.|
|Authors:||Smyth, Mark J;Kershaw, M H;Hulett, M D;McKenzie, Ian F C;Trapani, Joseph A|
|Affiliation:||Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia.|
|Citation:||Journal of Leukocyte Biology; 55(4): 514-22|
|Abstract:||Expression of the gene encoding the cytolytic granule protein perforin is restricted to cytotoxic lymphocytes. To undertake a functional analysis of the immediate 5'-promoter region of the mouse perforin gene, we transiently transfected mouse perforin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs into cytotoxic T, T lymphoid, B-lymphoid, and nonlymphoid cell lines. The transcriptional activity of the perforin promoter was restricted to cytotoxic lymphocytes. The perforin promoter was controlled by several positive (in perforin-positive cells) and negative (in perforin-negative cells) cis-acting regions, spread over at least 1.1 kilobases. The most specific expression of the CAT reporter gene in the interleukin-2-dependent cytotoxic T cell line CTLL-R8 was obtained with the mouse perforin promoter encompassing positions -1104 to +1 in relation to the RNA cap site. This construct expressed 65- to 70-fold higher CAT activity than the promoterless CAT construct in perforin-expressing cells but only 1- to 5-fold higher CAT activity than the promoterless construct in nonlymphoid cells. On the basis of these data, we used this most specifically active mouse perforin promoter, -1104 to +1, to express in CTLL-R8, a chimeric human receptor comprising the extracellular domains of human Fc gamma RI and the transmembrane and intracellular domains of TCR zeta. Selection in G418-containing medium produced CTLL-R8 transfectant clones that (1) expressed high levels of human Fc gamma RI mRNA; (2) expressed cell surface Fc gamma RI as demonstrated by immunoprecipitation and their ability to bind the Fc portion of human and mouse monoclonal antibodies (mAbs) in an isotype-specific manner, and (3) bound RBC expressing mucin-1 (Muc-1) peptide in the presence of a chimeric mouse-human anti-Muc-1 mAb. Activation of CTLL-R8 transfectants upon engagement of the human Fc gamma RI was evidenced by their ability to lyse tumor target cells in an mAb isotype-dependent manner. The successful expression of a functional chimeric gene in CTLL-R8 suggests that the mouse perforin promoter represents a novel reagent for expressing exogenous genes in cytotoxic T lymphocytes.|
|Internal ID Number:||8145022|
Molecular Sequence Data
Pore Forming Cytotoxic Proteins
Promoter Regions, Genetic
|Appears in Collections:||Journal articles|
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