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.
1. Embryo microinjection
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 containing the embryos.
Figure 2: Co-axial controls and needle holder. A) The co-axial controls (arrow) allow for 3-dimensional movement of the needle to ensure precise placement of the needle for each injection. It is important to use co-axial controls to raise the needle vertically when switching slides so that the needle does not collide with the new slide when putting it into place on the stage. Lateral co-axial controls allow for accurate penetration of the needle into the posterior pole. B) Image of needle holder showing angle for injection.
Figure 3: Chamber for egg hatching. A,B) Carefully wash injected embryos into a cylindrical container filled to a quarter depth with double-distilled water and lined with filter paper. C,D) Movement of the container causes the eggs to naturally adhere to the filter paper. Before leaving the container for hatching, ensure any eggs that have adhered higher up the filter paper are gently washed back down to water level. The blue oval marks a number of deposited eggs to show relative size.
The authors have nothing to disclose.
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) |