Female Tigriopus californicus with egg sack. Photo by Morgan Kelly
Last week in his post “Transcriptomics in the wild (populations),” TME contributor Noah Snyder-Mackler focused on a recent paper by Alvarez et al. that reviews the last decade of transcriptomic research including the goal of linking gene expression and phenotype. Researchers today routinely collect transcriptomic data for non-model organisms but without robust genomic resources, (for example, a well-annotated genome) and/or the ability to perform genomic manipulations (for example, knockout organisms), it is often difficult (and sometimes controversial) to assign function to candidate genes.
The tide pool copepod Tigriopus californicus (pictured above) is an up and coming model system for a wide range of research areas including physiology, neurobiology, ecology, speciation, hybridization, and local adaptation. The Burton, Edmands, Kelly, and Willett labs (among others) continue to generate genomic and transcriptomic data for Tigriopus and a new method published recently in Molecular Ecology Resources by Barreto, Schoville and Burton is an important contribution to the Tigriopus genomic toolbox.
Barreto et al. development an RNA interference (RNAi) method that knocks out target genes in Tigriopus californicus.
RNAi is triggered by the delivery of gene-specific double-stranded RNA (dsRNA) to the cytoplasm. The dsRNA is cleaved into fragments of 21–25 nucleotides (small interfering RNAs, or siRNAs), which become associated with the multiprotein RNA-induced silencing complex (RISC). Based on sequence similarity, the RISC-siRNA structure then targets and degrades endogenous mRNAs before they can be used for translation into protein.
-Barreto et al. 2015
RNAi allows you to knock out a candidate gene, perhaps a gene previously identified as differentially expressed between treatments, but which lacks a good annotation, and determine how the loss of function of that gene affects the organism. This gets us closer to linking gene expression at a given locus to phenotype. Barreto et al. developed RNAi for heat-shock beta 1 (hspb1) based previous results that this gene has >100-fold upregulation in T. californicus during extreme heat stress (Schoville et al. 2012).
One of the tricky parts of RNAi is getting the double stranded RNA (dsRNA) fragments that will degrade the mRNA of the gene to be knocked out into the organism. Baretto et al. tested four methods in Tigriopus.
- Feeding: to put it simply, the authors mixed the dsRNA into the copepod’s food.
2. Lipofection: this technique inserts genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane. Barreto et al. incubated copepods in a solution that included lipid-based carriers which inserted the dsRNA into the copepod’s cells.
3. Rehydration: the copepods were incubated in hypersaline water (around 100ppt, 3X the strength of normal seawater) for 30-60 minutes- which dehydrates them but doesn’t kill them (these little crustaceans are tough!). Next the copepods were placed in a normal seawater solution containing the dsRNA. The idea here is that the dsRNA will be taken up by the copepods as osmotic balance is restored.
4. Electroporation: this is a molecular technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane. Electric current was passed through a test tube with copepods incubated in a solution containing dsRNA.
Gene knockdown for all methods was assessed by qPCR shortly (3-6 days) after each treatment and it was determined that electroporation was the most effective method of introducing the dsRNA into the copepods. Next, in a new set of experiments, the authors used electroporation to assess the time of onset and short-term duration of hspb1 knockdown. They found that the hspb1 dsRNA replicates were successful at knocking down expression of the heat shock gene, while controls had no effect on gene expression (Figure 1a below). Barreto et al. then exposed copepods treated with hspb1 dsDNA and control copepods to high temperature stress and those with knocked down hspb1 expression had the lowest survivorship (Figure 1b).
Fig. 1 Tolerance of Tigriopus californicus to high thermal stress during heat-shock beta 1 (hspb1) suppression. Copepods were electroporated in assay mixture containing hspb1 dsRNA (n = 10, red lines), nonspecific control dsRNA (n = 6, dashed blue lines) or no dsRNA (n = 6, solid blue lines). Four days after treatment, subsamples of copepods were used to quantify (a) expression of hsbp1 (relative to myosin and GAPDH). The remaining subsamples were exposed to a high temperature stress (36 °C for 1 h), and (b) their survivorship was monitored for 5 days. Figure and caption taken from Barreto et al.
Although these results are copepod-specific, RNAi has been used successfully in a range of taxa including cnidarians, sponges, flatworms, rotifers, tardigrades, gastropods, and arachnids (see references in Baretto et al. 2015). Combining tools like RNAi with RNAseq experiments in non-model and emerging model organisms gets us closer to our goal of linking gene expression and phenotype and furthers our understanding of functional pathways.
Alvarez, M., Schrey, A. W., & Richards, C. L. (early online). Ten years of transcriptomics in wild populations: what have we learned about their ecology and evolution? Molecular Ecology. DOI: 10.1111/mec.13055
Graur, D., Zheng, Y., Price, N., Azevedo, R. B., Zufall, R. A., & Elhaik, E. (2013). On the immortality of television sets: “function” in the human genome according to the evolution-free gospel of ENCODE. Genome Biology and Evolution, 5, 578-590. DOI: 10.1093/gbe/evt028
Barreto, F. S., Schoville, S. D., & Burton, R. S. (early online). Reverse genetics in the tide pool: knock‐down of target gene expression via RNA interference in the copepod Tigriopus californicus. Molecular Ecology Resources. DOI: 10.1111/1755-0998.12359
Schoville, S. D., Barreto, F. S., Moy, G. W., Wolff, A., & Burton, R. S. (2012). Investigating the molecular basis of local adaptation to thermal stress: population differences in gene expression across the transcriptome of the copepod Tigriopus californicus. BMC Evolutionary Biology, 12, 170. DOI: 10.1186/1471-2148-12-170