Flp Frt system another gift from Yeast to Fly

“Yeast” Saccharomyces cerevisiae has contributed immensely in making Drosophila melanogaster a successful model system.The over expression system of UAS GAL4, which is borrowed from Yeast turned out to be real boon for fly geneticists.Another significant contribution came in the form of FLP FRT technique. The system uses the 2um plasmid of the yeast Saccharomyces cerevisiae.This plasmid encodes a recombinase (FLP) which acts on copies of the FLP recombination target (FRT) present in the plasmid as 599 bp inverted repeats. Flp Frt system was first time introduced in fly by Kent G. Golic and Susan Lindquist in their Cell publication of 1989 and later Xu.T and Gerry Rubin modified it for performing systematic genetic screens in order to trace mutations affecting many biological processes.

Need for Somatic clones/Mitotic clones:

During development of an organism many genes are used multiple time at various stages and this makes its very hard to detect the effects when you study the null allele for that gene.The development and function of adult organs requires genetic input from a large number of genes. Many of these genes perform essential roles during early development and due to this essential role,animals that lack them often die at early developmental stages . Hence ruling out the possibility to study the contribution of that particular gene in the adult organ formation.The effect of these genes on development can be analyzed by generating clones of homozygous cells in an otherwise heterozygous animal. Here the Flp Frt system comes to rescue of Drosophila workers.

The transgenic FRT sites when present at identical positions are very well recognised by the FLP recombinase and this could be because homologue’s are paired in mitotic cells in Drosophila, in contrast to most other organisms.

System of FLP FRT: How it works?

Mitotic clones can be created by generating flies with transgenic FRT sites at identical positions on homologous chromosomes. If the site-specific recombination between homologues chromosomes (non sister chromatids)occurs, and the daughter chromatids segregate appropriately after replication, the region of the chromosome arm that lies distal to the FRT site will be made homozygous, with each daughter cell inheriting two copies of this region from one of the parental chromosomes,which can then be later screened for a phenotype.

In this way one cell end up with two copies of mutant allele of gene in study and another cell will have two copies of wild type version.Normally these FRT flies are marked by some reporter (e.g LACZ or GFP which expresses in all cells or adult cuticular markers) genes in order to mark the clones generated.In initial experiments flp enzyme was under control of heat shock (hs) promoter and now a days people use UAS flp or flp fused to promoter of choice(like eyeless) which gives an option of expressing in tissue of interest.So by hs or crossing to GAl4 flies induces somatic clones which can be screened by antibody staining for the gene or marker attached to FRT ( cells with two copies of mutant allele doesn’t stain for antibody but cells adjacent to them with two copies of wild type protein stains dark ,called twin spot).

A clever modification of this system by Chou and Perrimon has led to identification of genes which have maternal contribution(RNA’s for these proteins are deposited by mother in eggs).The refinement to the Flp/FRT system, called the dominant female sterile (DFS) technique, has been developed by to select for mutant clones in the germ line itself. The DFS method uses the dominant ovoD mutation to kill the non-recombinant germ cells, so that females lay only those eggs that are derived from homozygous mutant germ-line clones. This approach provides a powerful way to screen for the maternal-effect phenotypes of lethal genes.The two above techniques helped to uncover many genes involved in development of Drosophila which successfully managed to escape during many conventional genetic screens

Reference:

Golic KG, Lindquist S.
Cell. 1989 Nov 3;59(3):499-509.
The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome.

Xu T, Rubin GM.
Analysis of genetic mosaics in developing and adult Drosophila tissues.
Development. 1993 Apr;117(4):1223-37.

Chou TB, Perrimon N.
Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila.
Genetics. 1992 Jul;131(3):643-53.