An efficient transgenic system by TA cloning vectors and RNAi for C. elegans.
In the nematode, transgenic analyses have been performed by microinjection of DNA from various sources into the syncytium gonad. To expedite these transgenic analyses, we solved two potential problems in this work. First, we constructed an efficient TA-cloning vector system which is useful for any promoter. By amplifying the genomic DNA fragments which contain regulatory sequences with or without the coding region, we could easily construct plasmids expressing fluorescent protein fusion without considering restriction sites. We could dissect motor neurons with three colors in a single animal. Second, we used feeding RNAi to isolate transgenic strains which express lag-2::venus fusion gene. We found that the fusion protein is toxic when ectopically expressed in embryos but is functional to rescue a loss of function mutant in the lag-2 gene. Thus, the transgenic system described here should be useful to examine the protein function in the nematode.
Gengyo-Ando
Keiko
K
Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo 162-8666, Japan.
Yoshina
Sawako
S
Inoue
Hideshi
H
Mitani
Shohei
S
D000818:Animals; D030801:Animals, Genetically Modified / Q000502:physiology*; D017173:Caenorhabditis elegans / Q000502:physiology*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D034622:RNA Interference / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D029742:Caenorhabditis elegans Proteins; D011993:Recombinant Fusion Proteins