Daniel Wang

Wadim J. Kapulkin^{1, 2, 3}

1 Department of Virology and Immunopathology, National Institute of Public Health-PZH, Chocimska 24, 00-791 Warsaw, Poland
2 Department of Infectious Diseases, Microbiology and Parasitology, Faculty of Veterinary Medicine, Grochowska 272, 03-849 Warsaw, Poland
3 || Present address: Veterinary Consultancy, Conrada 01-922 Warsaw, Poland

Correspondence to: Wadim J. Kapulkin (wadim_kapulkin@yahoo.co.uk)

RNA-interference (Fire et al. 1998) is a popular ‘reverse-genetics’ screening strategy. In particular ingested variant of RNAi (Timmons et al. 1999) gained popularity for genome-wide screens, where bacterially expressed dsRNA is administrated per os utilizing modified plasmids and E. coli as a feeding vector (Timmons et al. 2001). Genome-wide RNAi screens are presently carried using RNAi feeding libraries.

Two types of genome-wide RNAi ‘feeding libraries’ entailing PCR-amplified _target regions are presently available: library of predicted gene-overlapping segments (Kamath et al. 2003) and amplified cDNA library (Rual et al. 2004). However, available genome-wide PCR-based recombinant RNAi libraries – resources consisting of dsRNA producing plasmids – depend heavily on gene predictions, hence the bias toward certain exon rich gene regions or certain cDNAs. Here, I report on a complementary resource facilitating an approach to RNAi screen relying on unbiased ‘forward-genetics’ strategy.

The experiments based on this approach started with the construction of the library of genomic segments incorporated into convergent T7 polymerase binding sites plasmid (PBS) vector (L4440 plasmid ), I ligated with standard, small scale liquid worm DNA prep digested with EcoRI and HindIII* and transformed into HT115(DE3) E. coli (Timmons et al. 2001). Resulting convergent T7 PBS library was estimated as ~95% recombinant. Convergent T7 library clones were fed individually with a standard protocol (as described previously, Kapulkin et al. 2005) into N2-derived worms, to confirm the apparent lethal/sterile phenotype occurs at a frequency of 1-2 per 24 clones tested.

Based on the above experiment, I think the forward RNA interference screening is useful and feasible, with strong expectation the presented screening mode will complement and extend on the existing, currently available, genome-wide RNAi resources.

*Other variants of the experiment explored elsewhere, involve the fragmented DNA ligated with other enzymes and/or linkers.

References

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998 Feb 19;391(6669):806-11.

Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, Welchman DP, Zipperlen P, Ahringer J. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature. 2003 Jan 16;421(6920):231-7.

Kapulkin WJ, Hiester BG, Link CD. Compensatory regulation among ER chaperones in C. elegans. FEBS Lett. 2005 Jun 6;579(14):3063-8. Erratum in: FEBS Lett. 2007 Dec 22;581(30):5952. Kapulkin, Vadim [corrected to Kapulkin, Wadim J].

Rual JF, Ceron J, Koreth J, Hao T, Nicot AS, Hirozane-Kishikawa T, Vandenhaute J, Orkin SH, Hill DE, van den Heuvel S, Vidal M. Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res. 2004 Oct;14(10B):2162-8.

Timmons L, Court DL, Fire A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene. 2001 Jan 24;263(1-2):103-12.

Timmons L, Fire A. Specific interference by ingested dsRNA. Nature. 1998 Oct 29;395(6705):854.

Laura L. Maurer¹, Chuanjia Jiang², Heileen Hsu-Kim², Joel N. Meyer¹

1 Nicholas School of the Environment, Duke University, Durham NC
2 Department of Civil & Environmental Engineering, Duke University, Durham NC

Correspondence to: Laura L. Maurer (laura.kubik@duke.edu)

Mitochondria are dynamic organelles that require a controlled balance of essential ions to perform critical functions within cells. The content of these elements can both influence the effect of and be influenced by different stressors, including aging (Klang et al., 2014) and exposure to toxic chemicals (Sahu et al., 2013). This article provides reference concentrations of five essential ions present in the mitochondrial fraction of young adult N2 C. elegans. Briefly, 50,000 N2s were grown to young adult stage for mitochondrial isolation. Mitochondria were isolated from whole nematodes by dounce homogenization and differential centrifugation into a crude mitochondrial fraction as described (Kayser et al., 2004) with minor modifications. Elemental analysis of mitochondrial fractions was performed by inductively coupled plasma mass spectrometry (ICP-MS). Samples were prepared by oven drying (95°C overnight) and HNO3 digestion (90°C for 4 h), followed by dilution with MilliQ water to a final HNO3 concentration of 3% prior to elemental analysis by ICP-MS. Results were normalized to protein content (BCA assay). Sodium (Na) was present in the highest concentrations compared to other elements (significantly higher than calcium (Ca), magnesium (Mg), iron (Fe), and manganese (Mn); One-way ANOVA with Tukey’s post-hoc test, *p≤0.05; means are denoted above each bar). Mitochondrial Na concentrations are significantly higher than Ca concentrations in mammalian cells (Murphy and Eisner, 2009), and mitochondria can be a sink for magnesium (Kubota et al., 2005) as well as iron in mammals. It should be noted, however, that measured mitochondrial ion concentrations are influenced greatly by the method used for isolation; caution should be exercised when interpreting relative ion concentrations. These data serve as reference concentrations for mitochondria isolated from young adult N2 utilizing the cited methods.

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References

Kayser, E.B., Morgan, P.G., and Sedensky, M.M. (2004). Mitochondrial complex I function affects halothane sensitivity in Caenorhabditis elegans. Anesthesiology 101, 365-372.

Klang, I.M., Schilling, B., Sorensen, D.J., Sahu, A.K., Kapahi, P., Andersen, J.K., Swoboda, P., Killilea, D.W., Gibson, B.W., and Lithgow, G.J. (2014). Iron promotes protein insolubility and aging in C. elegans. Aging (Albany NY) 6, 975-991.

Kubota, T., Shindo, Y., Tokuno, K., Komatsu, H., Ogawa, H., Kudo, S., Kitamura, Y., Suzuki, K., and Oka, K. (2005). Mitochondria are intracellular magnesium stores: investigation by simultaneous fluorescent imagings in PC12 cells. Biochim Biophys Acta 1744, 19-28.

Murphy, E., and Eisner, D.A. (2009). Regulation of intracellular and mitochondrial sodium in health and disease. Circ Res 104, 292-303.

Sahu, S.N., Lewis, J., Patel, I., Bozdag, S., Lee, J.H., Sprando, R., and Cinar, H.N. (2013). Genomic analysis of stress response against arsenic in Caenorhabditis elegans. PLoS One 8, e66431.

The WBG

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Forward and unbiased RNA-interference screen

References

Analysis of mitochondrial sodium, magnesium, calcium, manganese, and iron in wild-type (N2) C. elegans

Figures

References