Novel Approaches to High-throughput Measurements of Replicative Lifespan in Yeast by Microfluidic Size Sorting and Genetic Engineering
Saccharomyces cerevisiae has been well established as a model system in aging research for more than 50 years. Whereas the chronological life span (CLS) is defined as the length of time a non-dividing yeast cell survives, replicative lifespan (RLS) refers to the number of times an individual cell divides before it senesces. Substances influencing the RLS are of particular interest for the development of therapeutics that counteract early aging and cancer. The standard RLS assay in yeast is highly laborious, requiring separation of mother and daughter cells by micromanipulation after every division.
In cooperation with an SME in Berlin we are developing a high-throughput screen in a microfluidic device that enables to simultaneously test the influence of various drugs on the RLS of yeast. For this purpose, a drug sensitive yeast strain was established in the genetic background of the S. cerevisiae W303 strain via knockout of three main multidrug transporter. Cell size of mother and daughter cells was measured on a Zeiss fluorescence microscope, and data were evaluated with the prism software. Cells will be fixed via mechanical forces on a chip, and daughter cells will be monitored by a novel detection system.
In a second approach, a linearly growing yeast strain is generated via a daughter-specific selective knockout of cell cycle genes. Failure of the daughter cells to complete cell division should result in an accumulation of aging mother cells in liquid culture, providing a direct correlation between RLS and increase in optical density.
In cooperation with an SME in Berlin we are developing a high-throughput screen in a microfluidic device that enables to simultaneously test the influence of various drugs on the RLS of yeast. For this purpose, a drug sensitive yeast strain was established in the genetic background of the S. cerevisiae W303 strain via knockout of three main multidrug transporter. Cell size of mother and daughter cells was measured on a Zeiss fluorescence microscope, and data were evaluated with the prism software. Cells will be fixed via mechanical forces on a chip, and daughter cells will be monitored by a novel detection system.
In a second approach, a linearly growing yeast strain is generated via a daughter-specific selective knockout of cell cycle genes. Failure of the daughter cells to complete cell division should result in an accumulation of aging mother cells in liquid culture, providing a direct correlation between RLS and increase in optical density.
