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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Genetics

Microinjection Method for Anopheles gambiae Embryos

Published: July 7th, 2021

DOI:

10.3791/62591

1Department of Microbiology & Molecular Genetics, University of California, Irvine, 2Department of Molecular Biology & Biochemistry, University of California, Irvine

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.

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.

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 genomes2-11. 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....

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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 fem.......

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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.......

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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.......

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We are grateful to Drusilla Stillinger, Kiona Parker, Parrish Powell and Madeline Nottoli for mosquito husbandry. Funding was provided by the University of California, Irvine Malaria Initiative. AAJ is a Donald Bren Professor at the University of California, Irvine.

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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

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