RNAi-medieret Gene Knockdown og<em> In Vivo</em> Diurese Assay i Voksen Kvinde<em> Aedes aegypti</em> Myg
Summary
I denne protokol vi kombinerer RNAi-medieret gen silencing med en<em> In vivo</em> Diurese assay til at studere virkningerne knockdown af gener af interesse har på myg fluid udskillelse.
Abstract
Denne video protokol viser en effektiv teknik til at Knockdown et bestemt gen i et insekt og udføre en ny bioassay at måle udskillelse. Denne fremgangsmåde kan anvendes til at opnå en bedre forståelse af processen diurese i insekter, og er især nyttig ved undersøgelse af diurese i blod-fodring arthropoder, som er i stand til at optage store mængder væske i en enkelt blodmel.
Dette RNAi-medieret gen knockdown kombineret med en in vivo-diurese assay blev udviklet af Hansen laboratoriet at undersøge virkningerne af RNAi-medieret knockdown af aquaporin gener på Aedes aegypti myg diurese 1.
Protokollen er setup i to dele: den første demonstration viser, hvordan man kan konstruere en simpel myg injektionsanordning og hvordan man kan forberede og injicere dsRNA i thorax af myg for RNAi-medieret gen knockdown. Den anden demonstration viser, hvordan man fastlæggerudskillelseshastigheder i myg ved hjælp af et in vivo bioassay.
Protocol
Discussion
Det RNAi anvendte protokol er blevet udviklet i laboratoriet af Alexander Raikhel ved University of California Riverside 6,7 og svarer til en protokol udgivet af Garver og Dimopoulos 4. Den eksperimentelle fremgangsmåde er vist i denne video protokol kan anvendes til at studere gener involveret i insekt diurese i en in vivo omgivelser. De ekskretionsorganerne af insekter, de Malpighian tubuli, har tiltrukket sig interesse for generationer af forskere som en "simpel" mod…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne takker Victoria Carpenter for hendes kritiske kommentarer i denne protokol.
Materials
| Name of reagent or equipment | Company | Catalogue number | Comments |
| MEGAscript T7 High Yield Kit | Ambion, Inc. | AM1334 | |
| PBS buffer | Sigma-Aldrich | P4417 | |
| Plastic tubing | Local vendor | PVC | |
| 1 ml plastic pipette tip | VWR | 83007-376 | Blue tip |
| 1 ml syringe | Becton, Dickinson and Company | 309602 | |
| Scissors | Local vendor | ||
| Metal needle | Carolina Biologicals | 654307 | Size 5 |
| Fly pad | Genesee Scientific | 789060 | |
| Battery-powered aspirator w/ collection vial | UPMA Labs | IPMM 2000 | |
| Fine tip forceps | World Precision Instruments | 14095 | |
| Glass capillary needles | World Precision Instruments | 1B200-6 | |
| Stereo dissection microscope | Leica Microsystems | S6D | |
| Analytical precision balance | Mettler Toledo | AB54S | |
| Sucrose | Sigma-Aldrich | 84097 | |
| One pint waxed lined cardboard cups | Local vendor | Manufactured soup cups | |
| Mesh net | Local vendor | plastic fly gauze |
References
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