Mosquito Embryo Microinjection: A Technique to Deliver Exogenous DNA into Embryo Yolk of Anopheles Gambiae to Generate Germline Mutations
Published: April 30, 2023
Abstract
Source: Carballar-Lejarazú, R. et al, Microinjection Method for Anopheles gambiae Embryos. J. Vis. Exp. (2021).
In this video, we demonstrate the delivery of gene drive construct into the embryo of Anopheles gambiae mosquito through embryo microinjection technique. This procedure allows for integration of delivered DNA construct, through homologous recombination, into the germline cells to generate transformed mosquito population.
Protocol
1. Embryo microinjection Use a micro-loader tip to fill the needles with 2 µL of DNA mixture (plasmid DNA at a concentration of 300-400 ng/µL. Insert the needle into the needle holder and connect the automated pressure pump tubing (Figure 1). Important: Align the needle so that it makes an angle of 15° with the plane of the slide (Figure 1). Open the needle tip by carefully touching the first egg of Anopheles…
Representative Results
Figure 1: Needle position during the microinjections. A) Wide-angle view of the injection stage. B,C) Position the needle so that the aligned embryos form an angle of 15° with the injection needle. Do not elevate the arm of the microinjection instrument that holds the needle significantly higher than the microscope stage to ensure proper angling between the needle and slide …
Açıklamalar
The authors have nothing to disclose.
Materials
| 10x Microinjection Buffer | – | – | 1 mM NaHPO4 buffer, pH 6.8, 50 mM KCl |
| Blotting membrane (Zeta-Probe GT Genomic Tested Blotting Membrane) | Bio-Rad | Neatly and straightly cut into 2×1 cm piece | |
| De-ionized or double-distilled water (ddH20) | Mili-Q | In a wash bottle | |
| Dissecting microscope | Leica | Leica MZ12 | For embryo alignment |
| Glass container | Pyrex | No. 3140 | 125 x 65 |
| Glass slide | Fisher Brand | No. 12-549-3 | 75×26 mm |
| Microinjector | Sutter Instrument | XenoWorks Digital Microinjector | |
| Microloader Pipette tips | Eppendorf | 20 µL microloader epT.I.P.S. | |
| Micromanipulator | Sutter Instrument | XenoWorks Micromanipulator | |
| Micropipette | Rainin | 20 µL | |
| Microscope | Leica | DM 1000 LED or M165 FC | For microinjection |
| Nylon mesh | |||
| Paint brush | Blick | No. 05831-7040 | Fine, size 4/0 |
| Petri dish | Plastic, (60×15 mm, 90×15 mm) |
Etiketler
Bu Makaleden Alıntı Yapın
Mosquito Embryo Microinjection: A Technique to Deliver Exogenous DNA into Embryo Yolk of Anopheles Gambiae to Generate Germline Mutations. J. Vis. Exp. (Pending Publication), e20988, doi: (2023).