Members of the Lab, Summer '00 (starting clockwise from upper right):
General description of research done in the Montgomery (M2) Lab
The Hughes Program at Macalester
Erin has been performing a ton of RNAi assays this summer. She has been microinjecting double-stranded RNAs (dsRNAs) into C. elegans and C. briggsae targeting a number of genes, including actin, par-3, and skn-1. She and Garett have then been working together to analyse the RNAi phenotypes of the affected embryos by staining them with several different cell-specific or stage-specific markers. These markers include Rh-phalloidin (stains f-actin) and antibodies against tubulin (which stains mitotic spindles), pharyngeal cells (mAb 3NB12), intestinal cells (mAb 126), muscle cells (mAb 5-6), and germ cells (anti-GLH-3). These studies will help us to figure out whether the function of genes such as par-3 and skn-1 are evolutionarily conserved between C. elegans and C. briggsae. Erin also worked in the lab last summer on another project (along with Jeff Norman) to test some of the length and sequence identity requirements for RNAi to work effectively in C. elegans and C. briggsae. She presented this work at the Midwest C. elegans Meeting (see abstract) held at the University of Minnesota in June '00. This work was also presented (by Jeff Norman) at the National Meeting of the Society of Developmental Biology held in Boulder, CO (also June '00). (See some results!)
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Garett has been perfecting techniques for antibody staining and in situ hybridization. He has been testing to see if the markers and antibodies listed above (which were developed for C. elegans) will recognize the same cells and tissue types in other nematode species such as C. briggsae and Cephalobus sp. Having cell and tissue-specific markers allows us to describe mutant phenotypes in more detail and find out e.g., if pharyngeal cells are completely missing in skn-1 (RNAi) C. briggsae. If the cells are missing in the mutants (due to an absence of staining by the pharyngeal-specific antibody 3NB12), but stain appropriately in the wild type, then we know that skn-1 function is important for correct development of the pharynx. Garett first joined the lab as part of the Hughes Science Institute last summer and has helped with RNAi soaking experiments over the past academic year. (See some results!)
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Sarah has been involved with two projects this summer. One project involves a completely different beast: the cute little Hawaiian squid Eupymna scolopes, which houses symbiotic luminescent bacteria in a light organ located on the underside of its ink sac. E. scolopes has been developed as an an animal model for the study of the initiation and development of symbiosis. We are currently testing whether RNAi will work to knockdown gene activity in newly hatched squidlets, which would aid in elucidating the function of symbiosis-specific genes. Sarah's second project involves the nematode Cephalobus. She will be using degenerate PCR to pull-out the skn-1 homolog from this interesting worm species, whose early development is quite radically different from that of C. elegans, but whose cell lineage is actually very similar. If Sarah is successful in identifying a skn-1 homolog from Cephalobus, we will follow up with studies designed to determine the its function through localization and knockdown experiments.
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Jeremy has been learning a number of molecular biological techniques, including PCR, bacterial vector cloning, and in vitro transcription. His project entails the building of a number of gene constructs to be used in RNAi and worm transformation experiments. Some of the constructs will use a heat shock promoter to drive expression of dsRNAs at different stages of the worm lifecycle to identify embryonic versus post-embryonic functions of the targeted genes.
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