Further, this virus is amenable to assays in a 96-well format wit

Further, this virus is amenable to assays in a 96-well format with excellent Z′-factors, can be used at very low infectious doses if required, has modest instrument requirements, and was successfully used to

MK-2206 price assess the effect of both antibodies and siRNAs directed against EBOV in a proof-of-concept study. Vero E6 (African green monkey kidney, ATCC CRL-1586) (Earley and Johnson, 1988) and 293 (human embryonic kidney) cells were maintained in Dulbecco’s modified Eagle medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS; Life Technologies), 2 mM l-glutamine (Q; Life Technologies), and 100 U/ml penicillin and 100 g/ml streptomycin (PS; Life Technologies) and grown at 37 °C with 5% CO2. An

additional transcriptional unit was inserted into a full-length clone plasmid (pAmp-rgEBOV) (Shabman et al., 2013) containing a cDNA copy of the EBOV genome (strain Mayinga, accession SB203580 in vitro number AF086833.2) using standard cloning techniques (Fig. 1A). The open reading frame for a codon-optimized Firefly luciferase (luc2, Promega) or eGFP was then inserted into this additional transcriptional unit. Detailed cloning strategies can be found as Supplementary material. Rescue was performed as previously described (Hoenen et al., 2012). Briefly, Vero cells were transfected with 250 ng of full-length clone plasmid and expression plasmids for the EBOV proteins NP (125 ng), VP35 (125 ng), VP30 (75 ng) and L (1000 ng) as well as T7 polymerase (125 ng). 24 h post transfection the medium was exchanged, and 7 days post-transfection 1 ml of supernatant was

passaged onto fresh Vero cells for growth of a virus stock. This stock was harvested upon development of CPE. The genomes of the rescued viruses were fully sequenced, with no unwanted mutations being identified. Stock titers were determined by CPE-based TCID50 assay (see below). Infections were performed as previously described (Hoenen et al., 2012). For time-course analysis of luciferase expression infections were performed in 6-well format on until ice, and the supernatant was removed and cells scraped into 1 ml cold PBS at the indicated time-points after shifting the cells to 37 °C. 400 μl of these cells were spun down for 5 min at 1000g and 4 °C, and the pellet was resuspended in 200 μl 1x passive lysis buffer (Promega). Luciferase activity was measured in a GloMax-Multi Microplate Multimode Reader (Promega) 10 min after adding 100 μl lysate (equivalent to approximately 200,000 cells) to an equal amount of BrightGlo (Promega) in an opaque white 96-well plate.

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