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Tools for Anopheles gambiae Transgenesis.

Volohonsky G, Terenzi O, Soichot J, Naujoks DA, Nolan T, Windbichler N, Kapps D, Smidler AL, Vittu A, Costa G, Steinert S, Levashina EA, Blandin SA, Marois E - G3 (Bethesda) (2015)

Bottom Line: Transgenesis in disease vector mosquitoes has existed since the 2000s but has remained limited by the delicate biology of these insects.This toolbox contributed to rendering transgenesis routine in this species and is now enabling the development of increasingly refined genetic manipulations such as targeted mutagenesis.Some of the reagents and procedures reported here are easily transferable to other nonmodel species, including other disease vector or agricultural pest insects.

View Article: PubMed Central - PubMed

Affiliation: INSERM U963, CNRS UPR9022, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, 67084 Strasbourg, France.

No MeSH data available.


Related in: MedlinePlus

Set-up for handling mosquito eggs following micro-injection. Aligned injected eggs are left undisturbed on the microscope slide, which is dipped in water as shown, in a square plastic box (10 cm side). Capillarity through the Whatman filter paper and nitrocellulose membrane keeps the eggs wet and larvae will spontaneously crawl into the water on hatching.
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fig1: Set-up for handling mosquito eggs following micro-injection. Aligned injected eggs are left undisturbed on the microscope slide, which is dipped in water as shown, in a square plastic box (10 cm side). Capillarity through the Whatman filter paper and nitrocellulose membrane keeps the eggs wet and larvae will spontaneously crawl into the water on hatching.

Mentions: A. gambiae mosquitoes were maintained in standard insectary conditions (28°, 75–80% humidity, 12-hr/12-hr light/dark cycle). Larvae were raised in deionized water and fed finely ground TetraMin fish food. Embryo microinjection was performed essentially as described (Fuchs et al. 2013; Pondeville et al. 2014). Freshly laid eggs were directly aligned against the edge of a nitrocellulose membrane kept wet with overlaying filter paper soaked with demineralized water. A mix of plasmids totaling 400 ng/µl of DNA (0, 1 mM NaHPO4 buffer pH 6.8, 5 mM KCl, 60 ng/µl helper plasmid, and generally 85 ng/µl of each of four distinct transgenesis plasmids) was injected under a Nikon Eclipse TE2000-S inverted microscope using an Eppendorf Femtojet injector and TransferMan NK2 micromanipulator. Injections were performed using the compensation pressure of the device, which was kept at 6000 hPa to promote a constant moderate flow of the DNA solution out of the quartz capillary. Microinjected eggs were left undisturbed on the injection slides, which were placed diagonally in a container with 1-cm-deep demineralized water, the part of the filter paper most distant from the eggs was dipped in water so that eggs remained wet by capillarity (Figure 1). Adult mosquitoes that survived microinjection were separated according to sex and crossed en masse to an excess of fresh wild-type adults. Neonate progeny larvae from several successive gonotrophic cycles were screened by spotting groups of 50–80 onto the wells of a 24-well teflon-coated diagnostic slide (Erie Scientific, Menzel GmbH, Braunschweig, Germany) under a Zeiss Axiovert 200M fluorescence microscope. When a fluorescent larva was detected, it was carefully isolated from the remainder larvae with the cut tip of a P200 pipette.


Tools for Anopheles gambiae Transgenesis.

Volohonsky G, Terenzi O, Soichot J, Naujoks DA, Nolan T, Windbichler N, Kapps D, Smidler AL, Vittu A, Costa G, Steinert S, Levashina EA, Blandin SA, Marois E - G3 (Bethesda) (2015)

Set-up for handling mosquito eggs following micro-injection. Aligned injected eggs are left undisturbed on the microscope slide, which is dipped in water as shown, in a square plastic box (10 cm side). Capillarity through the Whatman filter paper and nitrocellulose membrane keeps the eggs wet and larvae will spontaneously crawl into the water on hatching.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4478545&req=5

fig1: Set-up for handling mosquito eggs following micro-injection. Aligned injected eggs are left undisturbed on the microscope slide, which is dipped in water as shown, in a square plastic box (10 cm side). Capillarity through the Whatman filter paper and nitrocellulose membrane keeps the eggs wet and larvae will spontaneously crawl into the water on hatching.
Mentions: A. gambiae mosquitoes were maintained in standard insectary conditions (28°, 75–80% humidity, 12-hr/12-hr light/dark cycle). Larvae were raised in deionized water and fed finely ground TetraMin fish food. Embryo microinjection was performed essentially as described (Fuchs et al. 2013; Pondeville et al. 2014). Freshly laid eggs were directly aligned against the edge of a nitrocellulose membrane kept wet with overlaying filter paper soaked with demineralized water. A mix of plasmids totaling 400 ng/µl of DNA (0, 1 mM NaHPO4 buffer pH 6.8, 5 mM KCl, 60 ng/µl helper plasmid, and generally 85 ng/µl of each of four distinct transgenesis plasmids) was injected under a Nikon Eclipse TE2000-S inverted microscope using an Eppendorf Femtojet injector and TransferMan NK2 micromanipulator. Injections were performed using the compensation pressure of the device, which was kept at 6000 hPa to promote a constant moderate flow of the DNA solution out of the quartz capillary. Microinjected eggs were left undisturbed on the injection slides, which were placed diagonally in a container with 1-cm-deep demineralized water, the part of the filter paper most distant from the eggs was dipped in water so that eggs remained wet by capillarity (Figure 1). Adult mosquitoes that survived microinjection were separated according to sex and crossed en masse to an excess of fresh wild-type adults. Neonate progeny larvae from several successive gonotrophic cycles were screened by spotting groups of 50–80 onto the wells of a 24-well teflon-coated diagnostic slide (Erie Scientific, Menzel GmbH, Braunschweig, Germany) under a Zeiss Axiovert 200M fluorescence microscope. When a fluorescent larva was detected, it was carefully isolated from the remainder larvae with the cut tip of a P200 pipette.

Bottom Line: Transgenesis in disease vector mosquitoes has existed since the 2000s but has remained limited by the delicate biology of these insects.This toolbox contributed to rendering transgenesis routine in this species and is now enabling the development of increasingly refined genetic manipulations such as targeted mutagenesis.Some of the reagents and procedures reported here are easily transferable to other nonmodel species, including other disease vector or agricultural pest insects.

View Article: PubMed Central - PubMed

Affiliation: INSERM U963, CNRS UPR9022, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, 67084 Strasbourg, France.

No MeSH data available.


Related in: MedlinePlus