Use of cDNA subtraction and RNA interference screens in combination reveals genes required for germ-line development in Caenorhabditis elegans

Caenorhabditis elegans is an ideal organism for the study of the molecular basis of fundamental biological processes such as germline development, esp ecially because of availability of the whole genome sequence and applicabil ity of the RNA interference (RNAi) technique. To identify genes involved in germ-line development, we produced subtracted cDNA pools either enriched f or or deprived of the cDNAs from germ-line tissues. We then performed diffe rential hybridization on the high-density cDNA grid, on which about 7,600 n onoverlapping expressed sequence tag (EST) clones were spotted, to identify a set of genes specifically expressed in the germ line. One hundred and si xty-eight clones were then tested with the RNAi technique. Of these, 15 clo nes showed sterility with a variety of defects in germ-line development. Se ven of them led to the production of unfertilized eggs, because of defects in spermatogenesis (4 clones), or defects in the oocytes (3 clones). The ot her 8 clones led to failure of oogenesis, These failures were caused by ger m-line proliferation defect (Glp phenotype), meiotic arrest, and defects in sperm-oocyte switch (Mog phenotype) among others. These results demonstrat e the efficacy of the screening strategy using the EST library combined wit h the RNAi technique in C, elegans.