Genetic tools in Drosophila have been well developed and can be used for a variety of purposes. There are ways to explore genes that control lifespan (e.g., quantitative trait loci (QTLs), longevity screens, candidate genes), and to identify genes that change their expression patterns in response to a particular treatment and/or with age (e.g., enhancer traps, microarray analysis). There are ways in which to manipulate the cellular environment by altering gene expressions in a time-dependent and tissue-specific manner (e.g., candidate gene approach). Below is an overview of such methods; comprehensive review articles on genetic approaches in aging studies in Drosophila can be found elsewhere (Partridge and Pletcher, 2003; Helfand and Rogina, 2003; Poirier and Seroude, 2005).
Quantitative Trait Locus (QTL) approaches QTL is a method for associating variation in quantitative genetic traits with specific regions of the genome. Back-crosses and intercrosses can be used to find markers on a genomic map that correlate with variation in the quantitative trait. The amount of variation in the trait that is explained by each locus can be quantified, and this information can then be used to target specific areas of the genome for further investigation. Ultimately, the utility of the QTL approach depends on a detailed genomic map and the ability to characterize the functions of genes in the regions identified, making Drosophila an excellent organism for this technique. Moreover, the cross-breeding and inbreeding process is facilitated in Drosophila by the wide variety of lineages available.
In particular, the combination of candidate gene approaches (see below) and QTL can be a powerful tool for identifying genes responsible for quantitative variation in traits such as lifespan. However, it must be cautioned that QTL results may be specific to the environment and/or genetic background under which the experiment is conducted, and care should be used in generalizing the results.
Phenotype (longevity) screening This is a method for identifying genes affecting lifespan by observing phenotypes (i.e., longevity) of flies that are generated through random gene mutations. Identification of mutated genes can be simplified by using transposition for mutagenesis: the P-element transposon is a commonly used mutagen. P-elements are transposable elements that are widely used to make mutations and manipulate the genome. Often random insertion of transposons into genes or their regulatory elements decreases gene activity (Cooley et al., 1988). For example, the methuselah gene, which encodes G-protein-coupled receptor, was identified by this method; loss of function in this by a P-element transposon resulted in 40% increases in average lifespan (Lin et al., 1998).
Enhancer Traps With enhancer traps, detailed gene expression patterns in specific tissues or cells can be characterized. In enhancer traps, a promoter, usually a P-transposable element, is inserted into the genome together with a reporter gene such as lacZ and GAL4. Enhancer regulation is expected to affect the reporter gene as well as its usual targets, so expression patterns of the reporter gene can be characterized and taken as a proxy for expression patterns of genes nearby the insertion locus. Then the region around the insertion can be identified and studied further in order to pinpoint what nearby gene(s) may be involved in the expression pattern and may thus be relevant to the phenomenon of interest (see O'Kane, 1998 for details). The gene Indy (I'm Not Dead Yet), which is associated with extended lifespan, was originally identified with an enhancer trap (Rogina et al., 2000).
Microarray Analysis Microarray analysis is a method that makes use of gene chips to which thousands of different mRNAs can bind and be quantified. By using such chips to quantify mRNA levels in different tissues or in individuals under different treatments, tens or hundreds of specific genes which vary in relation to the tissue or treatment can be identified, aiding in a mechanistic understanding of the differences. Further work can then be conducted using candidate locus approaches (see below). In contrast to QTL, which looks at allelic variation, microarray analysis looks at gene regulation: potentially, but not necessarily, a result of allelic variation.
As with QTLs, care must be exercised not only to control for genetic background and environment, but also to limit interpretation of results to the genetic background and environment studied. Also, it must be remembered that microarray analysis is by its nature correlative, and that further study of patterns is generally necessary for clear interpretation. That said, microarray analysis remains one of the most powerful techniques for examining genetic processes underlying physiological variation.
Drosophila in particular are well suited to micro-array analysis (a) because the genome is relatively small, meaning that most of it can be analyzed with a single microarray and that potentially important patterns are less likely to be missed, and (b) because the functions of many genes have already been studied, facilitating interpretation of results. Microarray analysis has been used in Drosophila to characterize gene expression changes during dietary restriction and with aging (Pletcher et al., 2002).
Candidate gene approaches The major use of candidate gene approaches is to test specific genes selected based on educated guesses as to which genes affect the process in question. The genes of interest can be deleted, overexpressed, or induced, and phenotypes of the mutant flies are observed accordingly. Mutation methods usually involve P-element mediated transgenes. They can be used to introduce foreign DNA into the Drosophila genome. However, depending on its position in the genome, the expression of an inserted transgene may vary and the insertion can have considerable effects on lifespan. Therefore, methods that allow the experimental and the control groups to have a transgenic insert in the same genomic location are desirable (Partridge and Pletcher, 2003; Helfand and
Rogina, 2003; Venken and Bellen, 2005; Poirier and Seroude, 2005).
There are techniques for "inducible" mutation that allow conditional expression of genes in a time- and tissue-specific manner. In principle, gene expression is initiated upon application of the inducer. The "flip-out" system uses a heat pulse as the inducer. However, the heat pulse can itself affect lifespan, potentially complicating the results. Two other systems, the "gene-switch" and the "tet-on" systems, use antiprogestin, RU486, and tetracycline or its analogue deoxycycline respectively, to feed flies to initiate gene expression.
Was this article helpful?
When it comes to reducing wrinkles, you really have to take your needs seriously. There are a number of factors that play a role in the health and well being of your skin. It is often hard to understand how products work and why they may not work even if they promise to do so. If you are considering wrinkle creams and a regimen of facelifts, you may go broke in the process. Before you do all of that, consider going through a process of determine the very best solution for your needs.