Cargando…

Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA

BACKGROUND: Ticks are blood-sucking arthropods responsible for transmitting a wide variety of disease-causing agents, and constitute important public health threats globally. Ixodes scapularis is the primary vector of the Lyme disease agent in the eastern and central U.S. RNAi is a mechanism by whic...

Descripción completa

Detalles Bibliográficos
Autores principales: Karim, Shahid, Troiano, Emily, Mather, Thomas N
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2823657/
https://www.ncbi.nlm.nih.gov/pubmed/20074328
http://dx.doi.org/10.1186/1472-6750-10-1
_version_ 1782177656489377792
author Karim, Shahid
Troiano, Emily
Mather, Thomas N
author_facet Karim, Shahid
Troiano, Emily
Mather, Thomas N
author_sort Karim, Shahid
collection PubMed
description BACKGROUND: Ticks are blood-sucking arthropods responsible for transmitting a wide variety of disease-causing agents, and constitute important public health threats globally. Ixodes scapularis is the primary vector of the Lyme disease agent in the eastern and central U.S. RNAi is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. Here, we describe an optimized protocol for effectively suppressing gene expression in the egg and nymphal stages of I. scapularis by electroporation. RESULTS: The genes encoding the putative Phospholipase A(2 )(PLA(2)), cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin were targeted by delivering the dsRNA encoding the specific gene coding regions in the unfed nymphs. Silencing was measured using real time qRT-PCR. Electroporation as a mode of dsRNA delivery appears to be substantially efficient and less traumatic to the tick than dsRNA microinjection in the unfed nymphs. Using Cy3-labeled dsRNA to monitor the movement, electroporated dsRNA entered the nymphs and spread to salivary glands and other tissues. The significant disruption of β-actin and cytoplasmic Cystatin transcripts in tick eggs demonstrate the applicability of this technique. The PLA(2), cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin genes were also significantly silenced, suggesting that this method has the potential to introduce dsRNA in eggs and unfed nymphs. CONCLUSIONS: Our study demonstrates that electroporation can be used as a simple dsRNA delivery tool in assessing the functional role of tick genes in the vector-host interactions. This technique represents a novel approach for specific gene suppression in immature stages of ticks.
format Text
id pubmed-2823657
institution National Center for Biotechnology Information
language English
publishDate 2010
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-28236572010-02-18 Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA Karim, Shahid Troiano, Emily Mather, Thomas N BMC Biotechnol Methodology article BACKGROUND: Ticks are blood-sucking arthropods responsible for transmitting a wide variety of disease-causing agents, and constitute important public health threats globally. Ixodes scapularis is the primary vector of the Lyme disease agent in the eastern and central U.S. RNAi is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. Here, we describe an optimized protocol for effectively suppressing gene expression in the egg and nymphal stages of I. scapularis by electroporation. RESULTS: The genes encoding the putative Phospholipase A(2 )(PLA(2)), cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin were targeted by delivering the dsRNA encoding the specific gene coding regions in the unfed nymphs. Silencing was measured using real time qRT-PCR. Electroporation as a mode of dsRNA delivery appears to be substantially efficient and less traumatic to the tick than dsRNA microinjection in the unfed nymphs. Using Cy3-labeled dsRNA to monitor the movement, electroporated dsRNA entered the nymphs and spread to salivary glands and other tissues. The significant disruption of β-actin and cytoplasmic Cystatin transcripts in tick eggs demonstrate the applicability of this technique. The PLA(2), cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin genes were also significantly silenced, suggesting that this method has the potential to introduce dsRNA in eggs and unfed nymphs. CONCLUSIONS: Our study demonstrates that electroporation can be used as a simple dsRNA delivery tool in assessing the functional role of tick genes in the vector-host interactions. This technique represents a novel approach for specific gene suppression in immature stages of ticks. BioMed Central 2010-01-14 /pmc/articles/PMC2823657/ /pubmed/20074328 http://dx.doi.org/10.1186/1472-6750-10-1 Text en Copyright ©2010 Karim et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology article
Karim, Shahid
Troiano, Emily
Mather, Thomas N
Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA
title Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA
title_full Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA
title_fullStr Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA
title_full_unstemmed Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA
title_short Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA
title_sort functional genomics tool: gene silencing in ixodes scapularis eggs and nymphs by electroporated dsrna
topic Methodology article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2823657/
https://www.ncbi.nlm.nih.gov/pubmed/20074328
http://dx.doi.org/10.1186/1472-6750-10-1
work_keys_str_mv AT karimshahid functionalgenomicstoolgenesilencinginixodesscapulariseggsandnymphsbyelectroporateddsrna
AT troianoemily functionalgenomicstoolgenesilencinginixodesscapulariseggsandnymphsbyelectroporateddsrna
AT matherthomasn functionalgenomicstoolgenesilencinginixodesscapulariseggsandnymphsbyelectroporateddsrna