2024-03-19T11:35:57Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1154212017-10-10T12:23:57Zcom_10261_9676com_10261_8col_10261_9677
2015-05-19T12:07:02Z
urn:hdl:10261/115421
Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: Evidence from Manduca sexta and Blattella germanica
Garbutt, Jennie S.
Bellés, Xavier
Richards, Elaine H.
Reynolds, Stuart E.
European Commission
Ministerio de Ciencia e Innovación (España)
Consejo Superior de Investigaciones Científicas (España)
Department for Environment, Food & Rural Affairs (UK)
Insect
Nuclease
Virus
dsRNA
RNAi
RNA interference (RNAi) is a specific gene silencing mechanism mediated by double-stranded RNA (dsRNA), which has been harnessed as a useful reverse genetics tool in insects. Unfortunately, however, this technology has been limited by the variable sensitivity of insect species to RNAi. We propose that rapid degradation of dsRNA in insect hemolymph could impede gene silencing by RNAi and experimentally investigate the dynamics of dsRNA persistence in two insects, the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. An ex vivo assay revealed that dsRNA was rapidly degraded by an enzyme in M. sexta hemolymph plasma, whilst dsRNA persisted much longer in B. germanica plasma. A quantitative reverse transcription PCR-based assay revealed that dsRNA, accordingly, disappeared rapidly from M. sexta hemolymph in vivo. The M. sexta dsRNAse is inactivated by exposure to high temperature and is inhibited by EDTA. These findings lead us to propose that the rate of persistence of dsRNA in insect hemolymph (mediated by the action of one or more nucleases) could be an important factor in determining the susceptibility of insect species to RNAi. © 2012 Elsevier Ltd.
2015-05-19T12:07:02Z
2015-05-19T12:07:02Z
2013-02
2015-05-19T12:07:03Z
artículo
Journal of Insect Physiology 59(2): 171-178 (2013)
http://hdl.handle.net/10261/115421
10.1016/j.jinsphys.2012.05.013
http://dx.doi.org/10.13039/501100000780
http://dx.doi.org/10.13039/501100004837
http://dx.doi.org/10.13039/501100003339
http://dx.doi.org/10.13039/501100000277
eng
http://dx.doi.org/10.1016/j.jinsphys.2012.05.013
Sí
closedAccess
Elsevier