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Characterization of human Fc?RI? chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines.

ABSTRACT: Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (Fc?RIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of Fc?RIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected Fc?RIH chain in humanized RBL cell lines. We compared surface levels of Fc?RI?H by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. Fc?RI?H copy numbers were assessed by qPCR, and the sequence verified. Transfection with Fc?RI?H cDNA was assessed for its ability to increase Fc?RI?H expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower Fc?RI?H expression, respectively. This was neither related to Fc?RIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, Fc?RI?H surface expression appeared to correlate with the co-expression of Fc?RI?H. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFc?RI gamma, which constitutively expresses Fc?RI?H, increased Fc?RI?H chain expression levels. Levels of Fc?RI?H surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.


PROVIDER: S-EPMC6701790 | BioStudies | 2019-01-01

REPOSITORIES: biostudies

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