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Title 

Development of an in vitro bioassay system for human thrombopoietin by constructing a recombinant murine cell line expressing human thrombopoietin receptor

Authors 

Heung Rok ParkHyo Jeong Hong

Publisher 

Springer Verlag (Germany)

Issue Date 

1997

Citation 

Molecules and Cells, vol. 7, no. 6, pp. 699-704

Keywords 

coloring agentcytokine receptorMPL protein, humanoncoproteinrecombinant proteintetrazoliumthiazole derivativethiazolyl bluethrombopoietinthrombopoietin receptor

Abstract 

We have developed a simple and rapid in vitro bioassay system for human thrombopoietin (hTPO) by constructing a recombinant murine BaF3 cell line expressing the hTPO receptor. The cDNA encoding hTPO receptor (c-Mpl) was cloned from human erythroleukemia (HEL) cells by reverse transcription-polymerase chain reaction (RT-PCR) and linked to the human cytomegalovirus promoter in pcDNA3 to yield expression plasmid phTR. The expression plasmid was stably transfected into BaF3 cells. The resulting transformants were initially selected in RPMI medium containing G418 and murine IL-3 (MuIL-3) and subjected to positive selection in the medium containing hTPO. Finally, cell proliferation of the selected clones in response to hTPO was measured using a colorimetric MTT assay. Most transformants showed a dose-dependent proliferation in response to 0.1 to 100 ng/ml hTPO, among them a cell clone (BaF-mpl), that showed a saturation density of 1.0 × 106 cells/ml and a doubling time of 16 h in the log growth phase. This clone was chosen for further characterization of hTPO-dependent proliferation. The BaF-mpl cells showed specificity for TPO, and they died within 24 h in the absence of TPO, which enabled us to complete the assay within 2 days. In addition, optimal MTT assay conditions were established for MTT treatment time and the number of cells to be added in the assay.

ISSN 

1016-8478

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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