Screen of neuronal genes by ectopic expression of cDNA in C.elegans

行为突变体的筛选和分析是鉴定神经系统功能和/或发育相关基因的有效途径。然而，通过常规的活性突变体筛选，其破坏导致致命性的基因，或存在于基因组中的冗余基因不能被识别。在这项研究中，我试图通过在线虫体内建立异位表达系统来鉴定新的神经元基因。为此，我构建了在神经元或普遍存在的启动子/增强子下表达的线虫cDNA文库，并将其引入线虫动物体内，检测了转基因线虫的表型。利用负责组织特异性表达的基因组片段构建了两个带有EcoRI-Noti定向克隆位点的表达载体。为了在神经元组织中驱动表达，H20是由Takeshi Ishihara博士在…的NIG通过启动子捕获而分离的基因组片段更多的日本人，被使用。为了在无处不在的组织中表达，使用了EF1a，它是日本九州大学的Makoto Koga博士分离的秀丽线虫延伸因子1a基因上游的3kb基因组片段。将从混合期虫体中分离的Poly(A)RNA合成的cDNA插入载体的定向克隆位点，构建由6×10^5个单克隆组成的文库。从每个由大约250个克隆组成的cDNA池中提取的质粒DNA与转化标记pRF4一起注射到成年两性动物的远端手臂中。受体ST2是一个转基因品系，其整个神经系统通过GFP的表达可视化。对于每个库，对独立建立的转化系的10多个F2或F3后代在解剖显微镜下观察其整体形态和运动，并在荧光显微镜下观察神经系统的形态。每个文库检查了3000个克隆。含有H20的文库中没有克隆导致F2代出现异常。从带有EF1a的文库中分离到一个带有9号池的F1转化子，产生了20条F2虫，其中16条是异常的；它们在L3期在推测的外阴区有突起，然后一瘸一拐地出现脱水。它们在没有产卵的情况下死去。我已经两次注射相同的池，但没有F1或F2转化子显示出类似的缺陷。较少

The screen and analysis of behavioral mutants has been a fruitful approach in identifying genes involved in function and/or development of nervous system. However, genes whose disruption lead to the lethality, or genes which are present redundantly in genome could not be identified by the conventional screen for viable mutants. In this study, I have attempted to identify novel neuronal genes by establishing an ectopic expression system in a worm, C.elegans. For this purpose, I have constructed C.elegans cDNA libraries which are expressed under a neuronal or an ubiquitous promoter/enhancer, have introduced the libraries into C.elegans animals, and examined the phenotype of the transgenic worms.Two expression vectors with an EcoRI-NotI directional cloning site have been constructed using genomic fragments responsible for the tissue specific expression. To drive expression in neuronal tissues, H20, a genomic fragment which was isolated by promoter trapping by Dr.Takeshi Ishihara at NIG in … More Japan, was used. For expression in ubiquitous tissues, EF1a, which is a 3kb genomic fragment upstream to the C.elegans elongation factor 1a gene isolated by Dr.Makoto Koga at Kyushu University in Japan, was used. cDNAs synthesized from poly (A) + RNA isolated from mixed-stage worms were inserted into the directional cloning site of the vectors, and libraries consisting of 6 x 10^5 individual clones were constructed. Plasmid DNA extracted from cDNA pools each consisting of around 250 clones were injected together with a transformation marker, pRF4, into the distal arms of adult hermaphrodites. The recipient ST2 is a transgenic strain whose entire nervous system was visualized by the expression of GFP.For each pool, more than 10 F2 or F3 progenies of independently established transformant lines were examined for their overall morphology and movement under a dissection microscope, and for the morphology of the nervous system under a fluorescent microscope.Three thousands clones have been examined for each libraries. No clone from the library with H20 caused an abnormality at F2 progenies. A F1 transformant with pool #9 from the library with EF1a gave rise to 20 F2 worms, of which 16 were abnormal ; they had protrusion at the presumptive vulval region at L3 stages, and then later limped and appeared dehydrated. They died without laying eggs. I have injected the same pool two more times, but no F1 or F2 transformants showed similar defects. Less