Under these conditions, the cultures were 95% homogeneous for neurons. selleck chemicals llc Neurons were cul tured for 7 days at 37 C in a humidified atmosphere containing 5% CO2, and suspended in serum free DMEM F12, for 24h prior to the treatments. Generation of a HIV 1 based lentiviral vector containing an expression cassette for a human soluble TNF a receptor Fc fusion protein A transfer plasmid containing an expression cassette for sTNFR Fc fusion protein was constructed. Briefly, a human codon optimized gene encoding the sTNFR Fc fusion protein was commercially synthesized. This gene contained the extracellular domain of the human TNF receptor type 2 fused through its carboxyl terminal to the hinge domain from the human IgG1 gene and the Fc domain from the human IgG3 gene.

The synthetic gene was then amplified by PCR, using primers containing Xho I and Sac II restriction sites within the 5 and 3 termini, respectively, and inserted Inhibitors,Modulators,Libraries into the pHR HB7 IRES GFP plasmid that was digested with Inhibitors,Modulators,Libraries the same enzymes. The final bicistronic plasmid construct, pHR hTNFR Fc eGFP, expressed the sTNFR Fc fusion protein and the green fluorescent protein. A DNA fragment encoding the hinge domain from human IgG1 and the Fc domain from human IgG3 was also amplified similarly through PCR and cloned into the same lentiviral vector plasmid through Xho I and Sac II digestion and ligation, and used as a control without the TNF a receptor. Lentiviral vec tors encoding the sTNFR Fc fusion protein, or the Fc fragment alone, were then produced by transient trans fection of 293T cells.

Vector production, Inhibitors,Modulators,Libraries concentration, and titration were performed as described, except that 293T cells were used for vector titration and initial detection of sTNFR Fc expression by western blot assay. Inhibitors,Modulators,Libraries Transduction of human microglial and neuroblastoma cells Briefly, 5 �� 105 HTB 11 or CHME 5 cells were suspended Inhibitors,Modulators,Libraries in 0. 4 mL RPMI 1640 med ium containing 8 ug mL polybrene in a 1. 5 mL tube, and 0. 1 mL of vector stock was added and incubated at 37 C for 2 h. Infected cells were then transferred into a 25 cm2 tissue culture flask with 2 mL of fresh growth medium and incubated at 37 C with 5% CO2. The medium was replaced 24 h post infection and transduction efficiencies were evalu ated on day 3 PI. The percentage of GFP positive cells was determined by calculating the number of GFP and total cells from randomly selected micro scopic fields under a fluorescence microscope.

A total of 3 microscopic fields, con taining at least 100 cells each, were counted for each transduction test. Western blotting The supernatant or lysate of transduced or non trans duced cells including 293T, HTB 11, and CHME 5 cells, was mixed with an equal volume of 2X sodium dodecyl sulfate sample buffer and loaded on 5% stack ing 12% separating SDS polyacrylamide toward gels.