Computational protocol: Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development

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Protocol publication

[…] Singled L4 staged hermaphrodites (n = 5) were placed on NGM plates seeded with Escherichia coli OP50 bacteria and the number of hatched embryos was counted over the next 4 days. Brood size was determined at 20°C, 25°C and 27°C.Synchronized L4 stage hermaphrodites were used for lifespan measurements. Worms were transferred every second day on NGM plates seeded with E. coli OP50 and examined every day for probe-provoked movement and pharyngeal pumping until death. Worms that died due to internally hatched eggs, desiccation or due to crawling out of the plate were censored. The cumulative survival rate was determined according to Kaplan and Meier (). The log-rank (Mantel–Cox) test was used for comparing significant distributions between different groups in the lifespan assays. All calculations and plots were performed with Microsoft Office Excel 2003, Octave 2.9.12 (version 2.x and 3.x,, Gnuplot (version 4.2, or R (version 2.7, [...] Mitochondrial morphology in the body wall muscle was examined by expressing GFP under the myo-3 promoter. Worms were mounted in four times diluted egg salts and observed with a Zeiss Axioplan 2 imaging microscope. All pixels representing mitochondria were annotated. The number of connected pixels was used as a measure of individual mitochondrial size. Image analysis was performed in ImageJ (ver. 1.40 and Matlab (ver.R2007, The MathWorks). [...] To localize nuclei in isolated gonads, Hoechst 33258 stain (1 µg/ml) was added to the mounting medium (M9) and the fluorescent staining was detected using a DAPI filter on a Zeiss Axioplan 2 Imaging microscope. After staining, the gonadal structure was analyzed with the software Endrov (ver.2.11.0) by 3D reconstruction of microscopic images and by annotation of stained nuclei. […]

Pipeline specifications

Software tools Gnuplot, ImageJ, Endrov
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Caenorhabditis elegans