Developing Highthroughput Methodologies For Lifespan Analysis

Until recently, discovery of genetic and environmental determinants of longevity in any organism has been driven largely by gene-specific studies based on prior knowledge or hypotheses. The development of new methods and technologies for moderate to high-throughput longevity phenotyping in yeast offers the promise of analyzing the replicative and chronological aging properties of thousands of strains simultaneously (see Chapter 10: Application of High-Throughput Technologies to Aging-Related Research). Recently, a method for high-throughput chronological life-span determination carried out in a 96-well format has been developed (T. Powers, M. Kaeberlein, B. Kennedy, S. Fields, unpublished data). This approach should provide the ability to carry out both large-scale genetic screens for mutations that alter chronological life span and screens for small molecules that alter the rate of aging in nondividing cells.

The ability to measure replicative life span in a high-throughput manner has been difficult to develop due to the nature of the replicative life-span assay, which requires the separation of daughter cells from mother cells (see The Replicative Life-Span Assay). Automated microdissection of daughter cells away from mother cells has proven difficult to implement for several reasons, and other methods of enriching for aged cells (for example, see Park et al., (2002)) are not sufficiently stringent to permit quantitative life-span determination. We have recently developed an iterative method that allows for moderate-throughput analysis of replicative life span for ~100 strains at one time (Kaeberlein and Kennedy, 2005). This is an improvement over past efforts; however, development of a true high-throughput method would be of immense value to the field.

The most obvious method by which high-throughput replicative life-span analysis might be accomplished is through the development of a system in which daughter cells are selectively killed. In theory, such a system could be engineered by the expression of a toxic product under the control of a daughter-specific promoter or by the expression of an essential product only in mother cells. In either case, the system would need to be inducible so that the strain can be propagated. The first system of this type to be developed uses the latter approach by expressing an essential gene, CDC6, under the control of the mother cell specific HO promoter (Jarolim et al., 2004). Unfortunately, this system appears to have serious flaws, as all three mutations observed to increase mother cell survival with this method fail to increase replicative life span as measured by the standard life-span assay (Jarolim et al., 2004). This may be due to a large amount of premature mother cell death, as evidenced by the fact that mother cell life span is shorted by about 70% when CDC6 is expressed from the HO promoter (Jarolim et al., 2004). Although not useful in its current form, this system represents a first step toward the development of a true high-throughput replicative life-span assay.

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