scholarly journals Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Raghuvir Viswanatha ◽  
Enzo Mameli ◽  
Jonathan Rodiger ◽  
Pierre Merckaert ◽  
Fabiana Feitosa-Suntheimer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Mosquito Species ◽  
Mosquito Cell ◽  
Exchange System ◽  
Public Health Burden ◽  
Cassette Exchange ◽  
Mosquito Cells ◽  
Mosquito Cell Lines ◽  
Genome Scale

AbstractMosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species.

scholarly journals Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

2021 ◽  
Author(s):  
Raghuvir Viswanatha ◽  
Enzo Mameli ◽  
Jonathan Rodiger ◽  
Pierre Merckaert ◽  
Fabiana Feitosa-Suntheimer ◽  
...  
Keyword(s):  
Public Health ◽  
Cell Lines ◽  
Mosquito Species ◽  
Screening Tools ◽  
Exchange System ◽  
Public Health Burden ◽  
Cassette Exchange ◽  
Health Burden ◽  
Mosquito Cell Lines ◽  
Genome Scale

Mosquito-borne diseases present a worldwide public health burden. Genome-scale screening tools that could inform our understanding of mosquitos and their control are lacking. Here, we adapt a recombination-mediated cassette exchange system for delivery of CRISPR sgRNA libraries into cell lines from several mosquito species and perform pooled CRISPR screens in an Anopheles cell line. To implement this method, we engineered modified mosquito cell lines, validated promoters and developed bioinformatics tools for multiple mosquito species.

scholarly journals Host Adaptation of a Wolbachia Strain after Long-Term Serial Passage in Mosquito Cell Lines

2008 ◽  
Vol 74 (22) ◽  
pp. 6963-6969 ◽  
Author(s):  
Conor J. McMeniman ◽  
Amanda M. Lane ◽  
Amy W. C. Fong ◽  
Denis A. Voronin ◽  
Iñaki Iturbe-Ormaetxe ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Mosquito Species ◽  
Fruit Fly ◽  
Serial Passage ◽  
Mosquito Cell ◽  
Original Strain ◽  
New Host ◽  
Mosquito Cell Lines

ABSTRACT The horizontal transfer of the bacterium Wolbachia pipientis between invertebrate hosts hinges on the ability of Wolbachia to adapt to new intracellular environments. The experimental transfer of Wolbachia between distantly related host species often results in the loss of infection, presumably due to an inability of Wolbachia to adapt quickly to the new host. To examine the process of adaptation to a novel host, we transferred a life-shortening Wolbachia strain, wMelPop, from the fruit fly Drosophila melanogaster into a cell line derived from the mosquito Aedes albopictus. After long-term serial passage in this cell line, we transferred the mosquito-adapted wMelPop into cell lines derived from two other mosquito species, Aedes aegypti and Anopheles gambiae. After a prolonged period of serial passage in mosquito cell lines, wMelPop was reintroduced into its native host, D. melanogaster, by embryonic microinjection. The cell line-adapted wMelPop strains were characterized by a loss of infectivity when reintroduced into the original host, grew to decreased densities, and had reduced abilities to cause life-shortening infection and cytoplasmic incompatibility compared to the original strain. We interpret these shifts in phenotype as evidence for genetic adaptation to the mosquito intracellular environment. The use of cell lines to preadapt Wolbachia to novel hosts is suggested as a possible strategy to improve the success of transinfection in novel target insect species.

scholarly journals Knockdown of piRNA pathway proteins results in enhanced Semliki Forest virus production in mosquito cells

2013 ◽  
Vol 94 (7) ◽  
pp. 1680-1689 ◽  
Author(s):  
Esther Schnettler ◽  
Claire L. Donald ◽  
Stacey Human ◽  
Mick Watson ◽  
Ricky W. C. Siu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Semliki Forest Virus ◽  
Virus Production ◽  
Antiviral Responses ◽  
Mosquito Cells ◽  
Mosquito Cell Lines ◽  
Arbovirus Infection ◽  
Pirna Pathway ◽  
Sirna Pathway

The exogenous siRNA pathway is important in restricting arbovirus infection in mosquitoes. Less is known about the role of the PIWI-interacting RNA pathway, or piRNA pathway, in antiviral responses. Viral piRNA-like molecules have recently been described following infection of mosquitoes and derived cell lines with several arboviruses. The piRNA pathway has thus been suggested to function as an additional small RNA-mediated antiviral response to the known infection-induced siRNA response. Here we show that piRNA-like molecules are produced following infection with the naturally mosquito-borne Semliki Forest virus in mosquito cell lines. We show that knockdown of piRNA pathway proteins enhances the replication of this arbovirus and defines the contribution of piRNA pathway effectors, thus characterizing the antiviral properties of the piRNA pathway. In conclusion, arbovirus infection can trigger the piRNA pathway in mosquito cells, and knockdown of piRNA proteins enhances virus production.

scholarly journals Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the effects of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication

2019 ◽  
Vol 13 (11) ◽  
pp. e0007346 ◽  
Author(s):  
Anthony C. Fredericks ◽  
Tiffany A. Russell ◽  
Louisa E. Wallace ◽  
Andrew D. Davidson ◽  
Ana Fernandez-Sesma ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Cell Lines ◽  
Persistent Infection ◽  
Mosquito Cell ◽  
Mosquito Cell Lines

scholarly journals Mosquito Small RNA Responses to West Nile and Insect-Specific Virus Infections in Aedes and Culex Mosquito Cells

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 271 ◽  
Author(s):  
Giel Göertz ◽  
Pascal Miesen ◽  
Gijs Overheul ◽  
Ronald van Rij ◽  
Monique van Oers ◽  
...  
Keyword(s):  
Small Rna ◽  
De Novo ◽  
Mosquito Species ◽  
Acute Infection ◽  
Mosquito Cell ◽  
Flock House Virus ◽  
Virus Infections ◽  
West Nile ◽  
Mosquito Cell Lines ◽  
Small Rna Deep Sequencing

Small RNA mediated responses are essential for antiviral defence in mosquitoes, however, they appear to differ per virus-vector combination. To further investigate the diversity of small RNA responses against viruses in mosquitoes, we applied a small RNA deep sequencing approach on five mosquito cell lines: Culex tarsalis CT cells, Aedes albopictus U4.4 and C6/36 cells, Ae. aegypti Aag2 cells (cleared from cell fusing agent virus and Culex Y virus (CYV) by repetitive dsRNA transfections) and Ae. pseudoscutellaris AP-61 cells. De novo assembly of small RNAs revealed the presence of Phasi Charoen-like virus (PCLV), Calbertado virus, Flock House virus and a novel narnavirus in CT cells, CYV in U4.4 cells, and PCLV in Aag2 cells, whereas no insect-specific viruses (ISVs) were detected in C6/36 and AP-61 cells. Next, we investigated the small RNA responses to the identified ISVs and to acute infection with the arthropod-borne West Nile virus (WNV). We demonstrate that AP-61 and C6/36 cells do not produce siRNAs to WNV infection, suggesting that AP-61, like C6/36, are Dicer-2 deficient. CT cells produced a strong siRNA response to the persistent ISVs and acute WNV infection. Interestingly, CT cells also produced viral PIWI-interacting (pi)RNAs to PCLV, but not to WNV or any of the other ISVs. In contrast, in U4.4 and Aag2 cells, WNV siRNAs, and pi-like RNAs without typical ping-pong piRNA signature were observed, while this signature was present in PCLV piRNAs in Aag2 cells. Together, our results demonstrate that mosquito small RNA responses are strongly dependent on both the mosquito cell type and/or the mosquito species and family of the infecting virus.

Stable transformation of mosquito cell lines using a hsp70::neo fusion gene

Gene ◽  
1993 ◽  
Vol 136 (1-2) ◽  
pp. 129-136 ◽  
Author(s):  
Gareth J. Lycett ◽  
Julian M. Crampton
Keyword(s):  
Cell Lines ◽  
Fusion Gene ◽  
Stable Transformation ◽  
Mosquito Cell ◽  
Mosquito Cell Lines
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