Establishment of a Stable Cell Line Expressing Mouse Leptin Receptor (OB-Rb) in Human Embryonic Kidney 293 Cell and Preadipocyte 3T3L-1 Cell

Abstract

Leptin, the product of the obesity (ob) gene, is a multifunctional cytokine and a 16-kDa circulating hormone that primarily acts in the hypothalamus. It plays a key role in the regulation of food intake and energy expenditure. In addition, it has direct effects on many cell types on the periphery. Leptin acts through its transmembrane receptors (OB-Rb), which are the product of the leptin receptor (OB-R) gene. There are several alternative spliced isoforms (a-f). Only one of OB-R, which has the longest cytoplasmic domain, has full signaling capabilities and it is expressed in high levels in the hypothalamus. In the present study, we investigate the obligatory function of normal food intake and body weight regulation. In order to develop a cell model to screen the effects of bio-food materials regulating food intake and to research cancer, the leptin receptor (OB-Rb) gene from mouse brain total RNA was introduced into a retroviral vector (pLNCX2) designed for stably over-expressing OB-Rb. An ecotropic retroviral supernatant (pLNCX2-Ob-Rb) was added to 3T3-L1 (adipocyte cells) and HEK293 (human embryonic kidney cells). G418 was used as selection antibiotic for the establishment of cell lines that constitutively expressed the leptin receptor proteins. We assayed expression of the leptin receptor using western-blot and quantitative RT-PCR. (Cancer Prev Res 12, 240-245, 2007)

Key Words: Leptin, Leptin receptor (OB-Rb), Stable cell line, Retroviral vector, Infection