Use of cDNA subtraction and RNA interference screens in combination reveals genes required for germ-line development in Caenorhabditis elegans
Use of cDNA subtraction and RNA interference screens in combination reveals genes required for germ-line development in Caenorhabditis elegans
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DOI：
10.1073/pnas.141004698
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发表时间：
2001-07-17
影响因子：
11.1
通讯作者：
Iino, Y
中科院分区：
文献类型：
--
作者：
Hanazawa, M;Mochii, M;Iino, Y
秀丽隐杆线虫是研究种系发育等基本生物过程的分子基础的理想生物，特别是因为其全基因组序列的可用性和 RNA 干扰 (RNAi) 技术的适用性。为了鉴定参与种系发育的基因，我们产生了扣除的 cDNA 池，其中或者富集了种系组织中的 cDNA，或者缺失了来自种系组织的 cDNA。然后，我们在高密度 cDNA 网格上进行差异杂交，在网格上发现了约 7,600 个非重叠表达序列标签 (EST) 克隆，以鉴定一组在种系中特异性表达的基因。然后用 RNAi 技术测试了 168 个克隆。其中，15 个克隆表现出不育性，并在种系发育中存在多种缺陷。其中 7 个由于精子发生缺陷（4 个克隆）或卵母细胞缺陷（3 个克隆）而导致产生未受精卵。其他8个克隆导致卵子发生失败，这些失败是由种系增殖缺陷（Glp表型）、减数分裂停滞和精卵母细胞转换缺陷（Mog表型）等引起的。这些结果证明了在线虫中使用 EST 文库结合 RNAi 技术的筛选策略的有效性。
Caenorhabditis elegans is an ideal organism for the study of the molecular basis of fundamental biological processes such as germline 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.