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 system of rat and human. NPY has been proposed to play an important role in satiety. The aim of this study was to produce cell lines that secrete high levels of bioactive NPY. For this purpose, the complementary DNA [cDNA] that encodes NPY was isolated by PCR. The cDNA was then cloned into pCEP4, to form 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 IR-NPY 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