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Reverse genetics in the tide pool: knock‐down of target gene expression via RNA interference in the copepod T igriopus californicus
Author(s) -
Barreto Felipe S.,
Schoville Sean D.,
Burton Ronald S.
Publication year - 2015
Publication title -
molecular ecology resources
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.96
H-Index - 136
eISSN - 1755-0998
pISSN - 1755-098X
DOI - 10.1111/1755-0998.12359
Subject(s) - biology , rna interference , gene , reverse genetics , gene expression , genetics , phenotype , rna , genome
Reverse genetic tools are essential for characterizing phenotypes of novel genes and testing functional hypotheses generated from next‐generation sequencing studies. RNA interference ( RNA i) has been a widely used technique for describing or quantifying physiological, developmental or behavioural roles of target genes by suppressing their expression. The marine intertidal copepod T igriopus californicus has become an emerging model for evolutionary and physiological studies, but this species is not amenable to most genetic manipulation approaches. As crustaceans are susceptible to RNA i‐mediated gene knock‐down, we developed a simple method for delivery of gene‐specific double‐stranded RNA that results in significant suppression of target gene transcription levels. The protocol was examined on five genes of interest, and for each, at least 50% knock‐down in expression was achieved. While knock‐down levels did not reach 100% in any trial, a well‐controlled experiment with one heat‐shock gene showed unambiguously that such partial gene suppression may cause dramatic changes in phenotype. Copepods with suppressed expression of heat‐shock protein beta 1 ( hspb1 ) exhibited dramatically decreased tolerance to high temperatures, validating the importance of this gene during thermal stress, as proposed by a previous study. The application of this RNA i protocol in T . californicus will be invaluable for examining the role of genes putatively involved in reproductive isolation, mitochondrial function and local adaptation.