standard GAL4-UAS in Aedes aegypti

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Shengzhang Dong, Ph.D., Postdoctoral Researcher, Department of Veterinary Pathobiology, University of Missouri. MORE ABOUT THE AUTHOR

In a just-published paper Zhao et al. (2014) reported the development and testing of a gut-specific blood-driven carboxypeptidase (CP) Gal4 driver line of Ae. aegypti.

The CP-Gal4 driver was contained within a piggyBac transposable element and was composed of the 1.1 kb AeCP promotor sequence, the 0.8 kb chimeric Gal4 activator sequence, and a selectable marker EGFP gene driven by 3×P3 eye-specific promoter.

The GAL4-UAS system was first developed in Drosophila by Brand and Perrimon in 1993. This binary system is based on the properties of the yeast GAL4 transcription factor, which activates transcription of its target genes by binding to specific cis-regulatory sites called UAS (Upstream Activation Sequence).

Gal4/UAS Transcription System in Drosophila

Gal4/UAS Transcription System in Drosophila

The basic system is composed of two independent parent transgenic lines, the GAL4 driver line in which the Gal4 gene is expressed in a tissue-specific pattern and the UAS responder line in which the gene of interest is under UAS control.

Mating of the UAS-containing responder flies with the GAL4 driver-containing flies results in progeny bearing the two components, in which the UAS-transgene  is expressed in a transcriptional pattern that reflects that of the GAL4 driver.

Enhancer Trapping Technology in Anopheles stephensi - Trichogen/Tormogen Cells Expressing tdTomato

An example of the Gal4/UAS system being used in Anopheles stephensi.  Trichogen/Tormogen Cells Expressing GAL4 +UAS tdTomato
Photo Credit:  David O’Brochta, University of Maryland

Besides Drosophila, the GAL4-UAS system has been introduced into other insects such as Bombyx moriTribolium castaneum, Anopheles gambiae, Anopheles stephensi and Aedes aegypti.

In Ae. aegypti, the GAL4-UAS system was first reported by Kokoza and Raikhel in 2011 in which they developed two transgenic lines, a Vg-Gal4 driver and a UAS-EGFP responder line. After crossing the two lines, Vg-Gal4; UAS-EGFP hybrids expressed the reporter gene EGFP  in a tissue-, stage- and sex- specific pattern after blood feeding.

In the current paper by Zhao et al. (2014) the CP-Gal4 line was developed by germ-line transformation and had a specific fluorescent eye-color marker gene for easy identification. This line exhibited a tissue-, sex-, and stage-specific expression pattern similar to that of the endogenous CP gene.

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piggyBac vector with CP-Gal4 transgene.

After crossing the CP-Gal4 driver line with the UAS-EGFP reporter line, CP-Gal4; UAS-EGFP hybrid mosquitoes were produced and expressed EGFP only in the midgut after blood-feeding.

They also demonstrated that insulin/InR and nutrient-driven amino acid/ target-of-rapamycin pathways, but not 20E, were involved in regulating CP gene expression in CP-Gal4; UAS-EGFP lines.

Aedes aegypti.jpg

Aedes aegypti

The CP-Gal4 line is very important in that CP-Gal is only activated by blood feeding within two days. This CP-Gal4/UAS system will help to understand virus-vector interactions by investigating the functions of midgut-specific genes, especially those genes that are involved in or responsible for virus infection.

Zhao B, Kokoza VA, Saha TT, Wang S, Roy S, Raikhel AS (2014) Regulation of the gut-specific carboxypeptidase: a study using the binary Gal4/UAS system in the mosquito Aedes aegypti. Insect Biochem Mol Biol. 54:1-10. doi:10.1016/j.ibmb.2014.08.001

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