![]() We engineered VSV to express an eGFP–P fusion protein ( Fig. The VSV genome consists of a (–)-sense ssRNA molecule. The primers were as follows: VSV P/M forward primer, 5′-tcctgctcggcctgagatac-3′ VSV M/G forward primer, 5′-tcctggattctatcagccactt-3′ and VSV G/M reverse primer, 5′-ccagtttcctttttggttgtgt-3′. Background signal in RNA extracted from uninfected cells ranged from undetectable to 10 –4 of input RNA. VSV (–)-strand RNA concentrations were normalized to the VSV (–)-strand RNA concentration in an RNA extract of uninfected cells where virus had been added at moi = 3 during cell lysis for RNA extraction. ![]() To create a standard curve of VSV RNA concentrations, we made serial dilutions of RNA extracted from infected rde-1( 219) cells into RNA extracted from uninfected cells. elegans RNA-directed RNA polymerase activity, we designed the reverse transcriptase primer (primer VSV P/M forward) such that the cDNA would need to span both the P-M and M-G intergenic regions to produce a PCR product with the PCR primers used (primers VSV M/G forward and VSV G/M reverse). ![]() To minimize spurious detection of (–)-strand copies of viral transcripts produced by C. elegans cells, and the Qiagen Quantitect SYBR Green PCR kit for cDNA amplification. We used the Invitrogen ThermoScript reverse transcriptase kit to make cDNA from RNA extracted from infected C. elegans cell cultures by quantitative PCR of cDNA. We measured relative concentrations of VSV (–)-strand RNA in C. Uninfected controls were used in each experiment to correct GFP values for this background signal. No green fluorescence was visible by eye in uninfected cells, but background signal in the GFP channel due to stray light and fluorescence in the microscope optics (estimated by imaging an empty well) and background signal due to cellular autofluorescence (estimated by imaging uninfected cells by eye the autofluorescence is distinct from GFP due to the difference in color) were 11% and 8%, respectively, of the total captured light after a typical 7-day infection of wild-type cells at a multiplicity of infection (moi) of 3. The filter set used allows little or no detectable bleedthrough between fluorescence channels. To determine background signal for the Hoechst 33342 and GFP channels, we also collected data for unstained and uninfected cells, respectively. We set exposure times to avoid saturation of pixels by the brightest cells, such that the sum of the recorded image is linearly related to the amount of light reaching the camera. For each field, we focused on the nuclei in the Hoechst 33342 fluorescence channel, collected an image, and then collected an image blind from the GFP fluorescence channel. To measure relative amounts of GFP and DNA, we collected a linear transect of image pairs of 0.3- × 0.2-mm fields across each well. ![]() We imaged the cells in a widefield epifluorescence microscope mounted with a charge-couple device camera. Unless otherwise stated, we incubated cells at 27☌ for 7 days after infection, aspirated the medium, fixed the cells in 2% formaldehyde in PBS for 10 min at room temperature, rinsed once in PBS, stained for DNA for 10 min in PBS containing Hoechst 33342 dye, and then rinsed the cells two more times in PBS.
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