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 germ-line development, especially because of availability of the whole genome sequence and applicability of the RNA interference (RNAi) technique. To identify genes involved in germ-line development, we produced subtracted cDNA pools either enriched for or deprived of the cDNAs from germ-line tissues. We then performed differential hybridization on the high-density cDNA grid, on which about 7,600 nonoverlapping expressed sequence tag (EST) clones were spotted, to identify a set of genes specifically expressed in the germ line. One hundred and sixty-eight clones were then tested with the RNAi technique. Of these, 15 clones showed sterility with a variety of defects in germ-line development. Seven of them led to the production of unfertilized eggs, because of defects in spermatogenesis (4 clones), or defects in the oocytes (3 clones). The other 8 clones led to failure of oogenesis. These failures were caused by germ-line proliferation defect (Glp phenotype), meiotic arrest, and defects in sperm--oocyte switch (Mog phenotype) among others. These results demonstrate the efficacy of the screening strategy using the EST library combined with the RNAi technique in C. elegans.
Hanazawa
M
M
Molecular Genetics Research Laboratory, University of Tokyo, Tokyo 113-0033, Japan.
Mochii
M
M
Ueno
N
N
Kohara
Y
Y
Iino
Y
Y
D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D018076:DNA, Complementary; D018509:DNA, Helminth; D005306:Fertilization; D015870:Gene Expression; D017238:Genes, Helminth; D007246:Infertility; D009693:Nucleic Acid Hybridization / Q000379:methods; D009866:Oogenesis / Q000235:genetics* / Q000502:physiology; D010063:Ovum; D010641:Phenotype; D012313:RNA; D016372:RNA, Antisense / Q000235:genetics*; D018510:RNA, Helminth; D034741:RNA, Small Interfering; D013091:Spermatogenesis / Q000235:genetics* / Q000502:physiology
D018076:DNA, Complementary; D018509:DNA, Helminth; D016372:RNA, Antisense; D018510:RNA, Helminth; D034741:RNA, Small Interfering; D012313:RNA