Worm Breeder's Gazette 16(2): 29

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

A set of PCR primers for RNAi studies for 766 germ line genes

Valerie Reinke, Monica Colaiacovo, Gillian Stanfield, Anne M. Villeneuve, Stuart K. Kim

Department of Developmental Biology, Stanford University Medical Center, Stanford, CA 94305

In collaboration with Sam Ward's group, we have use DNA microarrays to identify genes with germ line enriched expression patterns. It should now be feasible to perform RNAi experiments to functionally deplete the activity of these genes and thereby gain insight regarding the genetic functions of the germ line on a whole-genome level. We plan to prepare a set of primers that can be used to generate PCR fragments suitable for RNAi experiments, and to distribute this set to the C. elegans germ line community.

We compared gene expression profiles in wild-type to those from a mutant with no germ line (glp-4(bn2)) to identify genes that were germ line-enriched. We also compared gene expression levels in a mutant making only sperm (fem-3(q23gf)) to those from a mutant making only oocytes (fem-1(hc17)) to identify sperm- and oocyte-enriched genes. We prepared polyA+ RNA from these strains, and then prepared either Cy3- or Cy5-labeled cDNA probes. We hybridized these probes to DNA microarrays containing 11,917 genes, representing 63% of the 19,099 predicted genes in the genome. The construction of these microarrays as well as details of the microarray experiment will be presented elsewhere. We performed each microarray experiment multiple times, and then identified genes that had reproducible differences in gene expression between the two samples (at the 99.9% confidence level using the Student's t-test). We identified 508 genes that were enriched in the germ line but expressed in both fem-3(gf) and fem-1(lf)(termed germ line-intrinsic ). We also identified 258 genes that were oocyte-enriched. These 766 genes may function in germ line stem cells, in meiosis, in recombination, in oogenesis or as maternal RNAs involved in embryonic patterning.

Having identified a large set of germ line-enriched genes, it is now feasible to determine the loss-of-function phenotype for each gene. We plan to interact with the C. elegans germ line community to collaboratively determine the effects caused by functional depletion using RNAi for each gene. We will prepare a set of primers for each gene that can be used in PCR experiments to amplify genomic DNA along with the sequence for the T7 RNA polymerase promoter. The PCR fragments can be used to produce dsRNA to be used in RNAi experiments. We are currently testing the feasibility of this approach by designing primers for a small number of positive control genes and showing that they can be used in RNAi experiments. Our preliminary results are promising, and we expect to have the full primer set available in the near future. Persons interested in obtaining a set of these primers should contact Valerie Reinke (reinke@cmgm.stanford.edu; 650-725-7611). Our hope is that results from RNAi experiments from different labs can be shared in a common database.