title: A recombineering pipeline for functional genomics applied to Caenorhabditis elegans.
abstract: 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.
authors.affiliation: Dresden University of Technology, BioInnovations Centre, Genomics Department, Tatzberg 47, 01307 Dresden, Germany.
authors.forename: Mihail
authors.initials: M
authors.lastname: Sarov
authors.forename: Susan
authors.initials: S
authors.lastname: Schneider
authors.forename: Andrei
authors.initials: A
authors.lastname: Pozniakovski
authors.forename: Assen
authors.initials: A
authors.lastname: Roguev
authors.forename: Susanne
authors.initials: S
authors.lastname: Ernst
authors.forename: Youming
authors.initials: Y
authors.lastname: Zhang
authors.forename: A Anthony
authors.initials: AA
authors.lastname: Hyman
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D023281:Genomics / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D010641:Phenotype; D034622:RNA Interference; D019076:Transgenes
chemical_list: D049452:Green Fluorescent Proteins