RESUMEN
Understanding the in vivo dynamics of protein localization and their physical interactions is important for many problems in biology. To enable systematic protein function interrogation in a multicellular context, we built a genome-scale transgenic platform for in vivo expression of fluorescent- and affinity-tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The platform combines computer-assisted transgene design, massively parallel DNA engineering, and next-generation sequencing to generate a resource of 14,637 genomic DNA transgenes, which covers 73% of the proteome. The multipurpose tag used allows any protein of interest to be localized in vivo or affinity purified using standard tag-based assays. We illustrate the utility of the resource by systematic chromatin immunopurification and automated 4D imaging, which produced detailed DNA binding and cell/tissue distribution maps for key transcription factor proteins.
Asunto(s)
Animales Modificados Genéticamente , Proteínas de Caenorhabditis elegans/análisis , Caenorhabditis elegans/genética , Ingeniería Genética/métodos , Genoma de los Helmintos , Factores de Transcripción/análisis , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Factores de Transcripción/genéticaRESUMEN
We present a new concept in DNA engineering based on a pipeline of serial recombineering steps in liquid culture. This approach is fast, straightforward and facilitates simultaneous processing of multiple samples in parallel. We validated the approach by generating green fluorescent protein (GFP)-tagged transgenes from Caenorhabditis briggsae genomic clones in a multistep pipeline that takes only 4 d. The transgenes were engineered with minimal disturbance to the natural genomic context so that the correct level and pattern of expression will be secured after transgenesis. An example transgene for the C. briggsae ortholog of lin-59 was used for ballistic transformation in Caenorhabditis elegans. We show that the cross-species transgene is correctly expressed and rescues RNA interference (RNAi)-mediated knockdown of the endogenous C. elegans gene. The strategy that we describe adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects.
Asunto(s)
Caenorhabditis elegans/genética , Ingeniería Genética/métodos , Genómica/métodos , Animales , Cromosomas Artificiales Bacterianos/genética , Clonación Molecular/métodos , Proteínas Fluorescentes Verdes/genética , Fenotipo , Interferencia de ARN , TransgenesRESUMEN
In cells lacking centrosomes, such as those found in female meiosis, chromosomes must nucleate and stabilize microtubules in order to form a bipolar spindle. Here we report the identification of Dasra A and Dasra B, two new components of the vertebrate chromosomal passenger complex containing Incenp, Survivin, and the kinase Aurora B, and demonstrate that this complex is required for chromatin-induced microtubule stabilization and spindle formation. The failure of microtubule stabilization caused by depletion of the chromosomal passenger complex was rescued by codepletion of the microtubule-depolymerizing kinesin MCAK, whose activity is negatively regulated by Aurora B. By contrast, we present evidence that the Ran-GTP pathway of chromatin-induced microtubule nucleation does not require the chromosomal passenger complex, indicating that the mechanisms of microtubule assembly by these two pathways are distinct. We propose that the chromosomal passenger complex regulates local MCAK activity to permit spindle formation via stabilization of chromatin-associated microtubules.