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

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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).

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