Mendenhall, Alexander et al. (2015) International Worm Meeting "Effects of introns on gene expression."

Mendenhall, Alexander, Brent, Roger, Sands, Bryan

[International Worm Meeting, 2015]

In C. elegans, as in the early days of recombinant DNA in mammalian cells, introns are generally used to increase transgene expression. Specifically, to increase expression, most C. elegans promoter-fused-to-fluorescent-protein reporter genes in use have three synthetic introns developed by the Fire lab inserted into the coding sequence of mCherry or GFP. But exactly how much more expression do those introns result in? Are all three introns necessary for the increased expression? Here, using single copy reporters integrated into the same defined locus, we measured the effects of different numbers, positions and sequences of introns on mean gene expression. We quantified expression from whole animals in flow and individual intestine cells using a point scanning confocal microscope. Preliminary findings suggest that, relative to no intron reporter gene controls, different combinations of the original three synthetic introns can result in increased or decreased mean expression. We intend to use these combinations to set reporter gene expression output to desired levels, for example, so as to be within the dynamic range of measurement of other reporters whose products are expressed in the same cells or in the same visual field. .

Mendenhall, Alexander et al. (2015) International Worm Meeting "Reproducible quantification of gene expression in single cells of live adult animals."

Tedesco, Patricia, Brent, Roger, Sands, Bryan, Mendenhall, Alexander, Johnson, Thomas

[International Worm Meeting, 2015]

In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. We developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. We hope they will allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to their molecular causes. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. We hope that understanding such variation will help reveal its contribution to differences in complex phenotypic outcomes in multicellular organisms.

Mendenhall, Alexander et al. (2015) International Worm Meeting "Mechanisms of animal-to-animal and cell-to-cell variation in gene expression in adult hermaphrodites."

Mendenhall, Alexander, Brent, Roger, Sands, Bryan, Johnson, Thomas, Tedesco, Patricia

[International Worm Meeting, 2015]

In isogenic populations of C. elegans cultured under homogeneous conditions, differences in the amount of GFP produced under control of the hsp-16.2 promoter predict differences in lifespan. Animals with high expression enjoy longer lifespan, longer healthspan and higher thermotolerance. How do animals end up in this privileged physiological state? We showed previously that variation (here, the coefficient of variation or CV) in hsp-16.2 reporter gene expression was constant among different strains bearing reporter constructs with differences in locus of integration, different copy numbers, and different fluorescent proteins. Here we sought to understand the origins of the variation in expression that results in high expression animals. We used a genetic and cell biological approach to study the origins of variable expression in individual animals and individual cells. Our preliminary results show that, at the level of the whole animal two distinct systems affect variation in gene expression, one of which increases, and the other of which decreases (canalizes) interindividual differences. At the cell level there is not much "intrinsic noise" in reporter expression (that is, fluorescent signal from two different differently colored reporter proteins driven by two different instances of the same promoter is highly correlated). In contrast to some studies in single cell organisms, we find that, when we measure the same cells in different animals, the ratio of fluorescent signal from two different fluorescent proteins driven by two different promoters is almost the same. It is possible that this limitation of variation in gene expression may reflect a need to tightly coordinate expression of different genes during metazoan development and adult life.