Transgenesis in Fruit fly, Drosophila melanogaster has come a long way from the days of landmark discovery of Spradling and Rubin in 1982. Drosophila research is highly dependent on P element-mediated transgenesis, as majority of its genetic tools depends on germline transformation by P- element transformation vector – like UAS-GAL4 system, UAS RNAi, FRT FLP for somatic and germ line clones to name some. This technique has two major drawbacks: the size of the DNA that can be integrated is limited and the insertion sites cannot be controlled.
P element has a tendency to integrate at the 5′ regulatory region of the gene thereby bringing it under unwanted regulation and adding to that integration of P element can disrupt the gene, which may or may be related to gene under study (e.g.if they are of same pathway). Sometimes depending upon the landing of p element the expression of gene is highly variable. Position effect of P element was bought under control by incorporating gypsy insulator but still it could also influence the gene it flanks.
This problem of random integration of P element during transgenesis was solved by making a modified transformation vector (pUAST is commonly used transformation vector in Drosophila) with attB sites from bacteriophage which recognizes attP sites available in fly (This will be a modified fly with attP sites to allow recombination with incoming attB plasmid which also has gene of interest to be over expressed) in presence of integrase source (A commonly used serine recombinase is the integrase from the Streptomyces bacteriophage phiC31.) In the case of attP/attB, new attL (att Left) and attR (att Right) sites are created , which are no longer substrates for the integrase, ensuring that recombination is irreversible.
Work from the lab of Hugo Bellen and Konrad Basler yielded flies with landing sites (attp) on all chromosomes along with the source of integrase which makes sure that the injected plasmid always lands at the same position in a particular fly.This modified vector also enables to clone large constructs sometimes upto 100KB. Later Norbert Perrimon’s lab have developed a transgenic RNA interference vector based on similar phiC31 site-specific integration method to overcome the disadvantages with random insertion of RNAi construct (like variability in the level of hairpin expression due to the random integration,where same construct at two different places behave differently depending upon place of integration)
References:
Venken, K. J., He, Y., Hoskins, R. A. and Bellen, H. J. (2006). P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.
Science 314, 1747-1751.
Bischof, J., Maeda, R. K., Hediger, M., Karch, F. and Basler, K. (2007). An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.
Proc. Natl. Acad. Sci. USA 104, 3312-3317.
Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster
Jian-Quan Ni, Michele Markstein, Richard Binari, Barret Pfeiffer, Lu-Ping Liu, Christians Villalta1, Matthew Booker,Lizabeth Perkins & Norbert Perrimon.
Nat Methods. 2008 Jan;5(1):49-51.
I have used these attP flies for making many transgenes and indeed the transformation efficiency is very good for some lines. But care has to be taken in ensuring the expression levels of gene to be induced. Some landing sites tend to express less than physiological levels of that particular gene, so one need to select a proper line (which express desired levels of protein) before starting off series of transgenes. These fly lines serves as a good system to compare various forms of a particular gene (e.g. point mutations) as they are inserted at same position. But lethality rate of embryos (after injection) are lot higher when compared to conventional transgenesis. Overall I prefer this system over conventional one when one has to compare many mutated versions of same protein.
Drosophila Image Credit : Smart Servier
Resource Link : Gompel lab protocol for generating transgenic flies – Injections of Drosophila embryos with intact chorion membrane