standard Anopheles gambiae Transgenesis Tool Box – Major Additions

Rob Harrell

Rob Harrell, Manager          University of Maryland’s Insect Transformation Facility,       Producing genetically modified insects for the research community, including Anopheles gambiae, An. stephensi and Aedes aegypti.

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Volohonsky et. al. (2015) in a just-published paper in G3 entitled  “Tools for Anopheles gambiae transgenesis”  describe a wealth of new tools for creating transgenic An. gambiae.

There has been a protocol for Anopheles gambiae transgenesis for over 15 years, however for most researchers the tools for functional genomics in Anopheles gambiae have been pretty sparse, that is until now.

Volohonsky et. al.’s tool kit includes:

  • 4 Anopheles gambiae PhiC31 docking site lines
  • 6 tissue specific fluorescent reporter lines
  • plasmids for efficient multiple-insert cloning
  • a puromycin-resistance cassette for transgenic larva selection
  • Cre recombinase-expressing line for loxP cassette excision
  • helper constructs for expressing:
    piggyBac transposase
    PhiC31 integrase
    Cre recombinase
  • a computer program for Anopheles gambiae codon-usage optimization for improving the expression of heterologous genes in An. gambiae.
AnophelesGambiaemosquito.jpg

Anopheles gambiae
Image by James D. Gathany
Transferred from en.wikipedia to Commons.The Public Health Image Library , ID#444. Licensed under Public Domain via Wikimedia Commons –

The PhiC31 docking strains Volohonsky et al created differ from previously described docking lines in that the genetic background of these lines is G3 while earlier lines were in the KIL strain.  They also differ in that the fluorescent markers used during creation of these lines have been removed by lox mediated cassette-exchange. The removal of the visible markers allows researchers to use donor constructs marked with any fluorescent marker. An added benefit of these lines lacking a visible marker is that up to 5 distinctly labeled donor constructs can be injected at once as a cocktail therefore streamlining both microinjection and screening.

integration reaction

Basic strategy of phiC31mediated transgene integration. Image from Calos Lab

The transgenesis vector kit includes a collection of GoldenGate cloning vectors, making relatively simple the task of assembling complex transgenic constructs. The vectors are pDSAT (mTurquoise) pDSAG (eGFP), pDSAY (EYFPvenus), pDSAR(DsRED), pDSAYN (YFPnls), pDSARN(DsREDnls), and pDSAP (Puromycinr).

Injecting Insect Embryos

Delivering genetic technologies to insect germ cells by microinjection of developing embryos.

The puromycin acety transferase (pac) marker allows researchers to streamline larval screening since wild-type larvae grown in the presence of puromycin die while transgenic larvae carrying the gene survive.

The authors state that all of the constructs have been proven to produce multiple transgenic lines. The vectors have been deposited in AddGene for distribution to the research community.

Cre recombinase-mediated excision

Another powerful tool in this kit is a Cre-recombinase expressing line that is on the third chromosome and marker-free. The authors point out that this line will allow the development of gene exchange systems in Anopheles gambiae like those developed for Drosophila and will open the door for making conditional knockouts similar to those used in mouse genetics.

This group also characterizes six useful promoter sequences: Prophenoloxidase gene (PPO6), Lipophorin (Lp), Vitellogenin (Vg) , Drosophila Actin5C and Anopheles gambiae G12.

PPO6 is expressed in hemocytes. Lp is expressed in fat body and approximately 12-48 hrs post blood meal the expression is further increase in females. Vg allows inducible expression of genes in female fat body approximately 18hours after a blood meal. In the midgut Drosophila Actin5C allows for constitutive expression and Anopheles gambiae G12 allows for inducible expression.

Lastly, the group introduces a computer program used to evaluate heterologous gene expression based on codon usage. This program should prove to be very useful in the re-engineering of promoters and genes from other species for use in Anopheles gambiae.

The technical resources reported here are significant and could prove to be very useful.  Delivery of these technologies to early embryos by microinjection and the maintenance of sizable collections of specialized lines remain significant challenges, but not insurmountable ones.

Tools for Anopheles gambiae Transgenesis (2015)  Gloria Volohonsky, Olivier Terenzi, Julien Soichot, Daniel A. Naujoks, Tony Nolan, Nikolai Windbichler, Delphine Kapps, Andrea L. Smidler, Anaïs Vittu, Giulia Costa, Stefanie Steinert, Elena A. Levashina, Stéphanie A. Blandin, and Eric Marois.  G3  Early Online April 13, 2015, doi:10.1534/g3.115.016808 

 

as of 5/11/15 – according to the authors “the supplementary information file on the G3 website has been updated, correcting a few sequencing mistakes in plasmid sequences”

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