Published: July 7th, 2021
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....
1. Preparing mosquitoes for microinjection
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.......
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.......
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.....
|10x Microinjection Buffer
|1 mM NaHPO4 buffer, pH 6.8, 50 mM KCl
|Blotting membrane (Zeta-Probe GT Genomic Tested Blotting Membrane)
|Neatly and straightly cut into 2x1 cm piece
|Conical tubes 50 ml (disposable centrifuge tube, polypropylene)
|De-ionized or double-distilled water (ddH20)
|In a wash bottle
|For embryo alignment
|No. 5 size
|125 x 65
|Model No. 150
|Latex dental film
|XenoWorks Digital Microinjector
|Microloader Pipette tips
|20 µL microloader epT.I.P.S.
|Sutter P-2000 micropipette puller
|DM 1000 LED or M165 FC
|Minimum fiber filter paper
|Chromatography Paper, Thick
|MR4, BEI Resources
|Anopheles gambiae, mated adult females, blood-fed 4-5 days post-eclosion
|1 mM, ph 6.8
|Fine, size 4/0
|Plastic, (60x15 mm, 90x15 mm)
|Quartz glass capillaries
|With filament, 1 mm OD, ID 0.7 10 cm length
|Water PCR grade
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