Mosaic (genetics) - Use in Experimental Biology

Use in Experimental Biology

Genetic mosaics can be extraordinarily useful in the study of biological systems, and can be created intentionally in many model organisms in a variety of ways. They often allow for the study of genes that are important for very early events in development, making it otherwise difficult to obtain adult organisms in which later effects would be apparent. Furthermore they can be used to determine the tissue or cell type in which a given gene is required and to determine whether a gene is cell autonomous. That is, whether or not the gene acts solely within the cell of that genotype, or if it affects neighboring cells which do not themselves contain that genotype, but take on that phenotype due to environmental differentiation.

The earliest examples of this involved transplantation experiments (technically creating chimeras) where cells from a blastula stage embryo from one genetic background are aspirated out and injected into a blastula stage embryo of a different genetic background.

Genetic mosaics are a particularly powerful tool when used in the commonly studied fruit fly, where they are created through mitotic recombination. Mosaics were originally created by irradiating flies heterozygous for a particular allele with X-rays, inducing double-strand DNA breaks which, when repaired, could result in a cell homozygous for one of the two alleles. After further rounds of replication, this cell would result in a patch, or "clone" of cells mutant for the allele being studied.

More recently the use of a transgene incorporated into the Drosophila genome has made the system far more flexible. The Flip Recombinase (or FLP) is a gene from the commonly studied yeast Saccharomyces cerevisiae which recognizes "Flip Recombinase Target" (FRT) sites, which are short sequences of DNA, and induces recombination between them. FRT sites have been inserted transgenically near the centromere of each chromosome arm of Drosophila melanogaster. The FLP gene can then be induced selectively, commonly using either the heat shock promoter or the GAL4/UAS system. The resulting clones can be identified either negatively or positively.

In negatively marked clones the fly is transheterozygous for a gene encoding a visible marker (commonly the green fluorescent protein, GFP) and an allele of a gene to be studied (both on chromosomes bearing FRT sites). After induction of FLP expression, cells that undergo recombination will have progeny that are homozygous for either the marker or the allele being studied. Therefore the cells that do not carry the marker (which are dark) can be identified as carrying a mutation.

It is sometimes inconvenient to use negatively marked clones, especially when generating very small patches of cells, where it is more difficult to see a dark spot on a bright background than a bright spot on a dark background. It is possible to create positively marked clones using the so called MARCM ("Mosaic Analysis with a Repressible Cell Marker", pronounced mark-em) system, developed by Liqun Luo, a professor at Stanford University and his post-doc, Tzumin Lee, now a group leader at Janelia Farm. This system builds on the GAL4/UAS system, which is used to express GFP in specific cells. However a globally expressed GAL80 gene is used to repress the action of GAL4, preventing the expression of GFP. Instead of using GFP to mark the wild type chromosome as above, GAL80 serves this purpose, so that when it is removed by mitotic recombination, GAL4 is allowed to function, and GFP turns on. This results in the cells of interest being marked brightly in a dark background.