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Reverse genetics in the tide pool: knock-down of target gene expression via RNA interference in the copepod Tigriopus californicus

Barreto, Felipe S. ; Schoville, Sean D. ; Burton, Ronald S.

Molecular ecology resources, 2015-07, Vol.15 (4), p.868-879 [Periódico revisado por pares]

England: Blackwell Publishing Ltd

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  • Título:
    Reverse genetics in the tide pool: knock-down of target gene expression via RNA interference in the copepod Tigriopus californicus
  • Autor: Barreto, Felipe S. ; Schoville, Sean D. ; Burton, Ronald S.
  • Assuntos: Animals ; copepod ; Copepoda ; Copepoda - genetics ; Gene Knockdown Techniques - methods ; gene suppression ; heat-shock protein ; Marine ; Molecular Sequence Data ; RNA Interference ; Sequence Analysis, DNA ; Tigriopus californicus
  • É parte de: Molecular ecology resources, 2015-07, Vol.15 (4), p.868-879
  • Notas: istex:E498D2F5E63293D74F4DBED1038FD6D3850490AB
    National Science Foundation - No. DEB1051057; No. IOS1155030
    Appendix S1 Assessment of expression stability of GAPDH and myosin for qPCR in Tigriopus californicus. Fig. S1 Quantification of GAPDH, myosin, and hspb1 expression in control and electroporated copepods.
    ark:/67375/WNG-D9VC647T-C
    ArticleID:MEN12359
    ObjectType-Article-1
    SourceType-Scholarly Journals-1
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  • Descrição: Reverse genetic tools are essential for characterizing phenotypes of novel genes and testing functional hypotheses generated from next‐generation sequencing studies. RNA interference (RNAi) 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 Tigriopus 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 RNAi‐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 RNAi protocol in T. californicus will be invaluable for examining the role of genes putatively involved in reproductive isolation, mitochondrial function and local adaptation.
  • Editor: England: Blackwell Publishing Ltd
  • Idioma: Inglês
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