A recombineering pipeline for functional genomics applied to Caenorhabditis elegans.
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.
Sarov
Mihail
M
Dresden University of Technology, BioInnovations Centre, Genomics Department, Tatzberg 47, 01307 Dresden, Germany.
Schneider
Susan
S
Pozniakovski
Andrei
A
Roguev
Assen
A
Ernst
Susanne
S
Zhang
Youming
Y
Hyman
A Anthony
AA
Stewart
A Francis
AF
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
D049452:Green Fluorescent Proteins