Drosophila melanogaster Germ‐line Transformation

Abstract

The ability to genetically transform and modify Drosophila melanogaster was originally made possible through the use of transposable elements (TEs). These proved to be efficient mutagens leading to the generation of many libraries containing TE‐tagged genes and enhancers throughout much of the genome. TEs were also efficient at introducing genes into this insect. A limitation of this technology was the inability to direct where transposons insert in the genome. However, recent advances made with the development of targeting systems based on protein‐based and RNA‐based guidance of endonucleases to specific target sites have provided a solution to this problem. As a consequence, the genetic toolbox of Drosophila geneticists have considerably expanded and will have a dramatic impact on our ability to understand genetic pathways in this insect.

Key Concepts:

  • Class II transposable elements form the basis of versatile genetic technology.

  • Homologous recombination technologies have been problematic but progress has been made.

  • Site‐specific recombinases add an additional dimension to the genetic toolbox.

  • Homologous recombination technologies have been problematic but progress has been made.

  • Zinc finger nucleases provide an alternate approach to site‐specificity.

  • TALENs facilitate another approach to directed integration into the genome.

  • CRISPRs will lead to a new generation of approaches to site‐specific medication in Drosophila.

Keywords: transposable element; homologous recombination; zinc finger nucleases; TALENs; CRISPR/Cas9

Figure 1.

Generating transgenic D. melanogaster. Recombinant plasmid DNA is injected into pre‐syncytial bastoderm embryos. The enzyme required for generating the transgenics is usually expressed from a helper plasmid. G0 flies are then backcrossed and the F1 generation is screened for expression of a dominant marker gene (M), such as GFP, the expression of which is commonly driven by an eye‐specific promoter (grey rectangle P), such as 3×3 P. Integration of foreign DNA can be achieved using different DNA cleaving enzymes. A binary or tertiary helper–donor system is used for all approaches. Transposable elements: Transposase expression is driven using a promoter from one plasmid (helper), while the other plasmid provides the transgene (donor), which is flanked by the transposable element's inverted terminal repeats (blue triangles). Upon integration into the host genome, the insertion site is duplicated (pale blue rectangles). Site‐specific recombinases: The ΦC31 viral recombinase can be used for site‐specific transgenesis. The donor plasmid containing the transgene and a recombination site (attB, yellow triangle) is injected together with a helper plasmid encoding the integrase into a transgenic fly strain that contains a docking site (yellow triangle). Recombination, mediated by the integrase between the recombination and docking sites leads to integration of transgene. DNA Recombinases: Most recently, the CRISPR system has been used to mediate site‐specific integration of a transgene. A donor plasmid containing the transgene and homology regions flanking the cleavage site (H5′ and H3′) is injected together with a helper plasmid encoding the Cas9 endonuclease, and two guide RNAs (red ribbons), which include 20‐nt of homology (blue line). The enzyme is guided to the target sites by two guide RNAs and, on cleavage of the target sites (black triangles), the transgene is introduced subsequently by homologous recombination.

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Pondeville E, Puchot N, Meredith JM et al. (2014) Efficient φC31 integrase–mediated site‐specific germline transformation of Anopheles gambiae. Nature Protocols 9(7): 1698–1712.

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How to Cite close
Atkinson, Peter W, and Michel, Kristin(Nov 2014) Drosophila melanogaster Germ‐line Transformation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002671.pub2]