The P[GAL4] enhancer-trap technique

The P-element transposon of Drosophila has provided us with an ideal mobilisable vector for the introduction of DNA-constructs, encoding a wide variety of genes under various control elements into the germ-line (Sentry and Kaiser, 1992, Finnegan, 1992). Wild-type P-elements are 2.9kb in length and include the gene for the transposase enzyme that facilitates transposition within the genome. This gene may be replaced with DNA encoding other genes of choice, and the transposase provided by other means (Robertson, et al., 1988).

This modified P-element technology enabled the development of enhancer-trap elements. An enhancer-trap contains a minimal promoter region, insufficient in itself, to induce transcription. If, after transposition, it inserts in a region under the influence of a local genomic enhancer, the inserted sequence (or reporter as it is known) should be transcribed in a pattern reflecting the enhancer activity. Initial enhancer-traps, P{lacZ} used the E.coli gene lacZ which encodes beta-galactosidase (beta-gal), enhancer activity then visualised simply by staining for beta-gal either through immunohistochemical techniques or more simply by using the chromogenic substrate for beta-gal, X-gal (O'Kane and Gehring, 1987). These 'first' generation enhancer-traps were of limited use in the study of neurons, due to the nuclear localisation signal attached to the reporter.

The 'second' generation P{GAL4} enhancer trap (see figure) works on a slightly more indirect principle (Brand and Perrimon, 1993). Rather than visualise the reporter directly, GAL4, a transcription factor from yeast that is functional in Drosophila (Fischer, et al., 1988) is expressed. GAL4 can be used to drive expression of any gene that is placed downstream of the activation signal, UASG. A construct with lacZ as a reporter (without the nuclear localisation signal) was developed (Brand and Perrimon, 1993). This allowed visualisation of cell bodies and, in neurons, axons and dendrites. In addition to allowing cell body visualisation, any DNA construct may be placed downstream of the GAL4 recognition site, thus the enhancer trap may be used to drive expression of cellular modifiers (Moffat, et al., 1992; Ferveur, et al., 1995; O'Dell, et al., 1995). A powerful approach to the visualisation of Drosophila brain anatomy using the P[GAL4] enhancer-trap technique has been recently described (Yang, et al., 1995). Using this technique, Yang et al. (1995) were able to selectively visualize the axonal processes of Mushroom Body (MB) intrinsic neurons, Kenyon cells (KCs), within the adult brain. They also described an unexpected, and otherwise covert, degree of anatomical subdivision within the MBs, based upon discrete patterns of reporter gene expression amongst subsets of KCs. The lines used in that particular study are described in this databse. In addition to their utility as markers for the adult brain, P[GAL4] enhancer-trap lines can be invaluable tools for describing brain development (Armstrong and Kaiser, 1997).

Key References

  • J. D. Armstrong and K. Kaiser (1997). The Study of Drosophila Brain Development. In L. M. Houdebine (Eds.), Transgenic animals - generation and use Harwood Academic Publishers.
  • A. Brand and N. Perrimon (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development, 118, pp 401-415.
  • J.-F. Ferveur, K. F. Stoertkuhl, R. F. Stocker and R. J. Greenspan (1995). Genetic feminisation of brain structures and changed sexual orientation in male Drosophila melanogaster. Science, 2647, pp 902-905.
  • D. J. Finnegan (1992). Transposable elements. Current Opinion in Genetics and Development, 2, pp 861-867.
  • J. A. Fischer, E. Giniger, T. Maniatis and M. Ptashne (1988). GAL4 activates transcription in Drosophila. Nature, 332, pp 853-856.
  • K. G. Moffat, J. H. Gould, H. K. Smith and C. J. O'Kane (1992). Inducable cell ablation in Drosophila by cold-sensitive ricin A chain. Development, 114, pp 681-687.
  • K. O'Dell, J. D. Armstrong, M. Y. Yang and K Kaiser (1995). Functional dissection of the Drosophila Mushroom Bodies by selective feminization of genetically defined subcompartments. Neuron. 15, pp 55-61
  • C. J. O'Kane and W. J. Gehring (1987). Detection in situ of genomic regulatory elements in Drosophila. Proc. Natl. Acad. Sci. USA, 84, pp 9123-9127.
  • H. M. Robertson, C. R. Preston, R. W. Phillis, D. M. Johnson-Schlitz, W. K. Benz and W. R. Engels (1988). A stable genomic source of P element transposase in Drosophila melanogaster. Genetics, 118, pp 461-470.
  • J. W. Sentry and K. Kaiser (1992). P-element transposition and targeted manipulation of the Drosophila genome. Trends in genetics, 8, pp 329-331.
  • M.-Y. Yang, J. D. Armstrong, I. Vilinsky, N. J. Strausfeld and K. Kaiser (1995). Subdivision of the Drosophila mushroom bodies by enhancer-trap expression patterns. Neuron, 15, pp 45-54.