Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1

Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.     

Length–weight relationships of two fish species from the Yangtze River,China

Fish samples for the study were collected from the Yangtze River in China. Length–weight relationships were determined for two endemic fish species (Leiocassis longirostris Günther, 1864 and Schistura fasciolata Nichols & Pope, 1927) for the first time.     

Intrathecal Administration of Tempol Reduces Chronic Constriction Injury-Induced Neuropathic Pain in Rats by Increasing SOD Activity and Inhibiting NGF Expression

We investigate the antinociceptive effect of intrathecal and intraperitoneal tempol administration in a rat model of chronic constriction injury (CCI)-induced neuropathic pain and explore the underlying antinociceptive mechanisms of tempol. Rats were randomly assigned to four groups (n = 8 per group): sham group, CCI group, Tem1 group (intrathecal injection of tempol), and Tem2 group (intraperitoneal injection of tempol). Neuropathic pain was induced by CCI of the sciatic nerve. Tempol was intrathecally or intraperitoneally administered daily for 7 days beginning on postoperative day one. The mechanical withdrawal threshold and thermal withdrawal latency were tested on preoperative day 3 and postoperative days 1, 3, 5, 7, 10, 14, and 21. Structural changes were examined by hematoxylin and eosin staining, toluidine blue staining, and electron microscopy. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels were determined using the thiobarbituric acid and nitroblue tetrazolium methods, respectively. Nerve growth factor (NGF) expression levels were determined by immunohistochemistry and Western blot. Intrathecal, but not intraperitoneal, injection of tempol produced a persistent antinociceptive effect. Intraperitoneal injection of tempol did not result in high enough concentration of tempol in the cerebrospinal fluid. Intrathecal, but not intraperitoneal, injection of tempol inhibited CCI-induced structural damage in the spinal cord reduced MDA levels, and increased SOD activities in the spinal cord. Furthermore, intrathecal, but not intraperitoneal, injection of tempol further downregulated the expression of NGF in the spinal cord following CCI, and this effect was blocked by p38MAPK inhibitor. Intrathecal injection of tempol produces antinociceptive effects and reduces CCI-induced structural damage in the spinal cord by increasing SOD activities and downregulating the expression of NGF via the p38MAPK pathway. Intraperitoneal administration of tempol does not exhibit antinociceptive effects.