Drosophila melanogaster Germ‐line Transformation


The ability to genetically transform and modify Drosophila melanogaster was originally made possible through the use of transposable elements (TEs).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.



Barrangou R, Fremaux C, Deveau H et al. (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819): 1709–1712.

Bellen HJ, Levis RW, He Y et al. (2011) The Drosophila gene disruption project: progress using transposons with distinctive site specificities. Genetics 188(3): 731–743.

Beumer KJ, Trautman JK, Christian M et al. (2013) Comparing zinc finger nucleases and transcription activator‐like effector nucleases for gene targeting in Drosophila. G3 3(10): 1717–1725.

Bibikova M, Golic M, Golic KG and Carroll D (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc‐finger nucleases. Genetics 161(3): 1169–1175.

Boch J, Scholze H, Schornack S et al. (2009) Breaking the code of DNA binding‐specificity of TAL‐Type III effectors. Science 326(5959): 1509–1512.

Cho SW, Kim S, Kim Y et al. (2014) Analysis of off‐target effects of CRISPR/Cas‐derived RNA‐guided endonucleases and nickases. Genome Research 24(1): 132–141.

Golic KG and Lindquist S (1989) The FLP recombinase of yeast catalyzes site‐specific recombination in the Drosophila genome. Cell 59(3): 499–509.

Gratz SJ, Cummings AM, Nguyen JN et al. (2013) Genome engineering of Drosophila with CRISPR RNA‐guided Cas9 nuclease. Genetics 194(4): 1029–1035.

Groth AC, Fish M, Nusse R et al. (2004) Construction of transgenic Drosophila by using the site‐specific integrase from the phage phiC31. Genetics 166(4): 1775–1782.

Jinek M, Chylinski K, Fonfara I and Calos MP (2012) A programmable dual‐RNA‐guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096): 816–821.

Linheiro RS and Bergman CM (2012) Whole genome sequencing reveals natural target site preferences of transposable elements in Drosophila melanogaster. PLoS ONE 7(2): e30008.

Liu J, Li C, Yu Z et al. (2012) Efficient and specific modifications of the Drosophila genome by means of an easy TALEN strategy. Journal of Genetics and Genomics 39(5): 209–215.

Metaxakis A, Oehler S, Klinakis A and Savakis B (2005) Minos as a genetic and genomic tool in Drosophila melanogaster. Genetics 171(2): 571–581.

Mitra R, Fain‐Thornton J and Craig NL (2008) piggyBac can bypass DNA synthesis during cut and paste transposition. EMBO Journal 27(7): 1097–1109.

Oberstein A, Pare A, Kaplan L and Small S (2005) Site specific transgenesis by Cre‐mediated recombination in Drosophila. Nature Methods 2(8): 583–585.

O'Brochta DA, Warren WD, Saville KJ and Atkinson PW (1996) Hermes, a functional non‐drosophilid gene vector from Musca domestica. Genetics 142(3): 907–914.

Rong YS and Golic KG (2000) Gene targeting by homologous recombination in Drosophila. Science 288(5473): 2013–2017.

Rong YS and Golic KG (2001) A targeted gene knockout in Drosophila. Genetics 157(3): 1307–1312.

Rubin GM and Spradling AC (1982) Genetic transformation of Drosophila with transposable element vectors. Science 218: 348–353.

Schornack S, Meyer A, Romer P, Jordan T and Lahaye T (2006) Gene‐for‐gene‐mediated recognition of nuclear‐targeted AvrBs3‐like bacterial effector proteins. Journal of Plant Physiology 163(3): 256–272.

Thibault ST, Singer MA, Miyazaki WY et al. (2004) A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nature Genetics 36(3): 283–287.

Urasaki A, Mito T, Noji S, Ueda R and Kawakami K (2008) Transposition of the vertebrate Tol2 transposable element in Drosophila melanogaster. Gene 425(1–2): 64–68.

Venken KJT, Schulze KL, Haelterman NA et al. (2011) MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes. Nature Methods 8(9): 737–743.

Zhang X, Ferreira IR and Schnorrer F (2014) A simple TALEN‐based protocol for efficient genome‐editing in Drosophila. Methods.

Further Reading

Barrangou R (2014) RNA events. Cas9 targeting and the CRISPR revolution. Science 344(6185): 707–708.

Charpentier E and Marraffini LA (2014) Harnessing CRISPR‐Cas9 immunity for genetic engineering. Current Opinion in Microbiology 19c: 114–119.

Craig N, Gragie R, Gellert M et al. (eds) (2002) Mobile DNA II, 2nd edn. Washington, DC: American Society for Microbiology.

Craig NL, Eickbush TH and Voytas DF (2010) Welcome to mobile DNA. Mobile DNA 1(1): 1–2.

O'Brochta DA (2014) Insect Genetic Resources Research Corodination Network. www.igtrcn.org

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.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

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]