Characterization of the conbercept gene localization in DHFR- amplified CHO cells using fluorescence in situ hybridization.

Chinese-hamster ovary (CHO) cells are most widely used for mammalian protein expression. After integration into the CHO genome, the exogenous gene may be lost in the process of large-scale protein production due to the removal of related selection pressures. Therefore, it is necessary to test its stability in the genome. Conbercept is a fusion protein that specifically binds to the various isoforms of vascular endothelial growth factor (VEGF)-A, VEGF-B, and placental growth factor (PlGF), thereby exerting anti-angiogenic activities. It has been approved for Phase Ⅲ clinical trials in the United States. In this study, fluorescence in situ hybridization (FISH) was used to localize the conbercept gene in dihydrofolatereductase (DHFR)-amplified CHO cell lines. Metaphase FISH showed that genomic integration of the conbercept gene was stable after 4 and 19 passages, and manifested three characteristics: first, the gene locates on one chromosome, rather than a number of chromosomes; second, the gene locates on the longer chromosomes; third, there are many copies located on the same chromosome. At the same time, the copy number of the conbercept gene in the CHO genome and the conbercept protein expression levels are also stable, as verified by qPCR and ELISA assays, respectively. These experiments demonstrated that the conbercept gene remained stable in the genome after 19 passages, and could be actively expressed, which strongly support the mass production and the quality control of conbercept.

In vivo anti-obesity effect of the agonist for receptor VPAC1.

It was hypothesized that the VPAC1 agonist may exert anti-obesity functions because VPAC1 is involved in the anorexigenic effects and the anti-inflammatory function of pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal polypeptide (VIP). Furthermore, our in vitro test showed that the expression of VPAC1 increased significantly after the 3T3-L1 adipocytes were differentiated, and that incubation of adipocytes with VPAC1 agonist (10-1 000 nmol/L per 1x10(6) cells) resulted in stimulation of lipolysis. To test the effect of VPAC1 agonist [Lys15, Arg16, Leu27]-VIP (1-7) GRF (8-27) on diet-induced obesity (DIO), we further designed the following two in vivo experiments: (1) Mice were fed on high-fat diet (HFD) and intraperitoneally (i.p.) treated with VPAC1 agonist simultaneously for 28 d; (2) Mice were given HFD for 35 d, and subsequently fed on the same HFD and i.p. treated with VPAC1 agonist for the next 28 d. The physiological indices, including body weight, weight of white adipose tissue, plasma glucose and blood lipid, were collected. The results showed that treatment with VPAC1 agonist inhibited ingestion significantly and prevented the elevations in body weight and the weights of the white adipose tissues (epididymal and dorsal) induced by HFD. The increases in plasma glucose, cholesterol, triglycerides and LDL induced by HFD were also down-regulated in mice treated with VPAC1 agonist. VPAC1 agonist treatment also improved the glucose tolerance. Therefore, VPAC1 agonist treatment inhibits the development of the obesity induced by HFD and helps to improve the morbidities associated with DIO.