Development of a pan-neuronal genetic driver in Aedes aegypti mosquitoes.
The recent development of neurogenetic tools in Aedes aegypti mosquitoes is beginning to shed light on the neural basis of behaviors that make this species a major vector of human disease. However, we still lack a pan-neuronal expression driver-a key tool that provides genetic access to all neurons. Here, we describe our efforts to fill this gap via CRISPR/Cas9-mediated knock-in of reporters to broadly expressed neural genes and report on the generation of two strains, a Syt1:GCaMP6s strain that expresses synaptically localized GCaMP and a brp-T2A-QF2w driver strain that can be used to drive and amplify expression of any effector via the Q binary system. Both manipulations broadly and uniformly label the nervous system with only modest effects on behavior. We expect these strains to facilitate neurobiological research in Ae. aegypti mosquitoes and document both successful and failed manipulations as a roadmap for similar tool development in other non-model species.
Pubmed ID: 34590074
Research resources used in this publication
- pQF2wWB (RRID:Addgene_61313) (plasmid)
- pQUAST-mCD8-GFP (RRID:Addgene_24351) (plasmid)
- Brp-T2A-QF2w-Hsp70-3XP3-dsRed (RRID:Addgene_141094) (plasmid)
- Syt1LeftArm-3XGS-GCaMP6s-SV40-3XP3-dsRed-SV40-Syt1RightArm (RRID:Addgene_159636) (plasmid)
- pBPGAL4.1Uw (RRID:Addgene_26226) (plasmid)
- pattB-synaptobrevin-7-QFBDAD-hsp70 (RRID:Addgene_46115) (plasmid)
- pGP-CMV-GCaMP6s (RRID:Addgene_40753) (plasmid)
Additional research tools detected in this publication
Antibodies used in this publication
- Bruchpilot antibody - Buchner, E.; Universitaetsklinikim Wuerzburg (RRID:AB_2314866)
- Cy3-AffiniPure Goat Anti-Mouse IgG (H+L) (min X Hu,Bov,Hrs Sr Prot) (RRID:AB_2338685)
- Goat Anti-Mouse IgG(H+L) Antibody, CF680 Conjugated (RRID:AB_10852820)
- Goat anti-Rabbit IgG (Heavy Chain), Superclonal Recombinant Secondary Antibody, Alexa Fluor™ 488 (RRID:AB_2536097)
- Rabbit Anti-Green Fluorescent Protein (GFP) Polyclonal Antibody, Unconjugated (RRID:AB_221569)
Associated grants
- Agency: NIAID NIH HHS, United States
Id: DP2 AI144246 - Agency: NIDCD NIH HHS, United States
Id: R00 DC012069
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This is a list of tools and resources that we have found mentioned in this publication.
Addgene (tool)
RRID:SCR_002037
Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
View all literature mentionsGENEWIZ (tool)
RRID:SCR_003177
Commercial organization for research and development genomics services and technical support to researchers.
View all literature mentionsQIAGEN (tool)
RRID:SCR_008539
A commercial organization which provides assay technologies to isolate DNA, RNA, and proteins from any biological sample. Assay technologies are then used to make specific target biomolecules, such as the DNA of a specific virus, visible for subsequent analysis.
View all literature mentionsBruchpilot antibody - Buchner, E.; Universitaetsklinikim Wuerzburg (antibody)
RRID:AB_2314866
This monoclonal targets Bruchpilot
View all literature mentionsCy3-AffiniPure Goat Anti-Mouse IgG (H+L) (min X Hu,Bov,Hrs Sr Prot) (antibody)
RRID:AB_2338685
This unknown targets Mouse IgG (H+L)
View all literature mentionsGoat Anti-Mouse IgG(H+L) Antibody, CF680 Conjugated (antibody)
RRID:AB_10852820
This unknown targets Goat Mouse IgG(H+L) CF680
View all literature mentionsGoat anti-Rabbit IgG (Heavy Chain), Superclonal Recombinant Secondary Antibody, Alexa Fluor™ 488 (antibody)
RRID:AB_2536097
This recombinant polyclonal secondary targets IgG (Heavy Chain)
View all literature mentionsRabbit Anti-Green Fluorescent Protein (GFP) Polyclonal Antibody, Unconjugated (antibody)
RRID:AB_221569
This polyclonal targets GFP
View all literature mentions
