Transfection of 6-23 a rat thyroid carcinoma cell line, with the Neuropeptide Y cDNA

Abstract

Neuropeptide Y (NPY) is a 36 amino acid peptide found throughout the central and peripheral nervous<br />system of rat and human. NPY has been proposed to play an important role in satiety. The aim of this study<br />was to produce cell lines that secrete high levels of bioactive NPY. For this purpose, the complementary<br />DNA (cDNA) that encodes NPY was isolated by PCR. The cDNA was then cloned into pCEP4, to form<br />pCEP4NPY. 6-23 cells were transfected with pCEP4NPY by electroporation. Transfected cells were selected by the addition of hygromycin B to the culture medium. Resistant colonies were picked and transferred to 96-well plates. The medium was tested for IRNPY using a specific NPY radioimmunoassay (RIA). The IR-NPY secreted by the cells was characterized by sephadex G50 chromatography and reversed phase fast protein liquid chromatography (FPLC). It was found to co-elute with the synthetic standard in both cases. RNA was extracted from the cells and subjected to Northern blot analysis using labeled NPY cDNA as a probe. The cells were found to express high levels of NPY at mRNA levels.