Creating focal genetic modifications in an intact animal is a powerful approach for studying the cellular interactions that underlie the development and function of tissues and organs. This method has been used to great advantage in Drosophila, facilitating the study of developmental questions relating to the autonomy of gene actions, restriction of cell fate and growth pattern of specific tissues. The approach involves the generation of genetic mosaics, tissues in which some cells differ from their neighbors by a single mutation, effecting either a gain or loss of function phenotype. Through the analysis of mutant and wild-type cells within mosaics patches, it is possible to draw inferences about interacting cells and in some cases the molecules and pathways subserving cellular communication.
The application of mosaic analysis to the study of nervous system function has the potential to yield a wealth of information because it should allow for an assessment of the function of particular gene products within individual cells that are part of a network. Thus, in tissues such as the nervous system where functional information resides not only in the nature and number of its constituent cells, but also in the manner in which they connect and temporally interact, it is essential that strategies be employed that neither unintentionally change the network nor eliminate some of the cellular constituents. Overall, the ideal strategy should permit stable genetic modification with precise temporal and spatial control.
Implicit in the use of genetic mosaic analysis is the ability to distinguish mutant from normal cells by the use of markers. The preferred marker is one which is gratuitous thus causing no cell damage, cell-autonomous so that cellular level resolution of mosaicism can be reliably scored, and having a short half-life to improve temporal analysis of the tissue after genetic modification.
Genetic mosaics have been generated in Drosophila by induction of mitotic recombination (reviewed in Ashburner, M., Drosophila: A Laboratory Handbook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is hereby incorporated by reference). Typically, radiation is used to induce DNA damage. In cells that have just completed DNA synthesis but have not yet divided, repair of the damage leads infrequently to homologous chromosome exchange. If the homologous chromosomes are appropriately marked, the resultant recombination event can be scored in daughter cells. More recently, high-frequency homologous chromosome exchange has been achieved by the use of the yeast FLP/FRT system (Golic K., Science, 252, 958 (1991); Golic, K. and Lindquist, S. Cell, 59, 499 (1989); Harrison, D. and Perrimon, N., Curr. Biol., 3, 424 (1993); Xu, T. and Rubin, G., Development, 117, 1223 (1993), which are hereby incorporated by reference). The FLP gene product is a site specific recombinase that catalyzes recombination at FRT target DNA elements. Expression in flies of FLP induces recombination between FRT elements on homologous chromosomes (Harrison, D. and Perrimon, N., Curr. Biol., 3, 424 (1993); Xu, T. and Rubin, G., Development, 117, 1223 (1993), which are hereby incorporated by reference). Inducible control of FLP by a heat shock promoter has been used in flies to grade the extent or recombination, thus modulating the extent of genetic mosaicism (Xu, T. and Rubin, G., Development, 117, 1223 (1993), which is hereby incorporated by reference).
Genetic mosaics in flies have also been generated by inducing intramolecular chromosomal recombination with the FLP/FRT system. In one example of this strategy, a transgene is constructed such that it is inactivated by the insertion of a DNA sequence encoding a stop codon flanked by FRT sites. After induction of FLP the inactivating DNA cassette is `flipped-out` allowing for the transcription of an mRNA that yields a translatable gene product (Struhl, G., Fitzgerald, K. and Greenwald, I., Cell, 73, 1323 (1993), which is hereby incorporated by reference). This approach could be used to generate gains or loss of function at any transcriptionally active transgene integration site or at a specific gene location targeted by homologous recombination.
Although the use of the FLP/FRT system in mammalian cells has been reported (O'Gorman, S., Fox, D. and Wahl, G., Science, 251, 1351 (1991), which is hereby incorporated by reference), a different recombination system, cre/loxP, has received wider attention and apparently greater success. The cre recombinase is bacteriophage P1-derived, and it interacts with its target site, loxP, a 34 bp element to produce site specific recombination (Sauer, B. and Henderson, N., Proc. Natl. Acad. Sci. USA 85, 5166 (1988); Sternberg, N. and Hamilton, D., J. Mol. Biol., 150, 467 (1981), which are hereby incorporated by reference). Using a binary approach in transgenic animals, investigators constructed and introduced separately into the germline of mice two different transgenes; the first, a strong promoter driving cre, and the second a recombinatorial substrate which constrained two loxP sites (Lasko, M., et al., Proc. Natl. Acad. Sci. USA, 89, 6232 (1992); Orban, P., Chiu, D. and Marth, J., Proc. Natl. Acad. Sci. USA, 89, 6861 (1992), which are hereby incorporated by reference). Crossing the two transgenic lines gave rise to compound heterozygotes in which recombination occurred in a highly efficient manner. The cre/loxP system has also been applied in embryonic stem (ES) cells to create deleted alleles by targeting a homologous locus with a construct that contains loxP elements flanking the region to be excised (Gu, H., Zou, Y.-R. and Rajewsky, K., Cell, 73, 1155 (1993), which is hereby incorporated by reference). With this approach the transient expression of cre produced a significant frequency of recombination events (Gu, H., Zou, Y.-R. and Rajewsky, K., Cell, 73, 1155 (1993), which is hereby incorporated by reference). Overall, the use of recombination systems in mice appears to satisfy the need for the efficient creation of stable genetic mosaics. However, the approach is significantly limited by constraints imposed by the characteristics of the promoter chosen to express the recombinase. Since every cell that expresses the recombinase will likely suffer a recombination event, it is difficult to use the system to generate mosaic tissues.
A need exists for a system to produce stable genetic mosaics where precise temporal and spatial control of gene expression can be obtained.