Microinjection Method for Anopheles gambiae Embryos

Summary

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

Abstract

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.

Introduction

Microinjection techniques have been used to experimentally manipulate organisms since the early 1900s¹. Microinjection has been used to study both basic biological functions and/or introduce important changes in the biology of a desired organism. The microinjection technique has been of particular interest to vector biologists and has been widely used to manipulate vector genomes^2-11. Transgenesis experiments in arthropod vectors often aim to make vectors less efficient at transmitting pathogens by either enacting changes that decrease a vector's fitness or increase refractoriness to the patho....

Protocol

1. Preparing mosquitoes for microinjection

1. Seed a cage¹³ (~5000 cm³) with ~100 male and 200-300 female 1-2 day adult post-eclosion mosquitoes and allow them to mate for 2 days.
2. After the mating period, provide mosquitoes a blood meal using either 2 mL of blood with an artificial feeding device or live anesthetized animals depending on insectary practices¹⁴. The following day provide mosquitoes a second blood meal to ensure that all mated females have had an opportunity to feed and that partially-fed mosquitoes have become fully-engorged.
3. Utilize the mosquitoes for embryo collect....

Results

A representative example of the application of the microinjection protocol described can be found in Carballar-Lejarazú et al⁵. The intent here was to insert an autonomous gene-drive system into the germline of a laboratory strain, G3, of An. gambiae. The system was designed to target the cardinal ortholog locus (Agcd) on the third chromosome in this species, which encodes a heme peroxidase that catalyzes the conversion of 3-hydroxykynurenine to xanthommatin, t.......

Discussion

With the increased availability of precise and flexible genetic engineering technologies such as CRISPR/Cas9, transgenic organisms can be developed in a more straightforward and stable way than previously possible. These tools have allowed researchers to create transgenic strains of mosquito vectors that are very close to achieving the desired properties of either refractoriness to pathogens (population modification) or heritable sterility (population suppression). However, to develop the most safe and stable genetically.......

Materials

Name	Company	Catalog Number	Comments
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 2x1 cm piece
Conical tubes 50 ml (disposable centrifuge tube, polypropylene)	Fisher Brand		Ends cut
De-ionized or double-distilled water (ddH20)	Mili-Q		In a wash bottle
Dissecting microscope	Leica	Leica MZ12	For embryo alignment
Forceps			No. 5 size
Glass container	Pyrex	No. 3140	125 x 65
Glass slide	Fisher Brand	No. 12-549-3	75x26 mm
Incubator	Barnsted Lab-line	Model No. 150	28 °C
KCl			50 mM
Latex dental film	Crosstex International	No. 19302
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
Micropipette puller	Sutter Instrument	Sutter P-2000 micropipette puller
Microscope	Leica	DM 1000 LED or M165 FC	For microinjection
Minimum fiber filter paper	Fisher Brand	No. 05-714-4	Chromatography Paper, Thick
Mosquitoes	MR4, BEI Resources		Anopheles gambiae, mated adult females, blood-fed 4-5 days post-eclosion
NaHPO4 buffer			1 mM, ph 6.8
Nylon mesh
Paint brush	Blick	No. 05831-7040	Fine, size 4/0
Petri dish			Plastic, (60x15 mm, 90x15 mm)
Sodium acetate			3M
Quartz glass capillaries	Sutter Instrument	No. QF100-70-10	With filament, 1 mm OD,  ID 0.7 10 cm length
Water PCR grade	Roche	No. 03315843001