A NEW VECTOR FOR CLONING LARGE EUKARYOTIC DNA SEGMENTS IN ESCHERICHIA-COLI

We investigated the relationship between plasmid size and electroporation efficiency in E. coli and found that even very large plasmids can be transfected efficiently. The efficiencies are well above the minimum required to construct representative libraries of complex eukaryotic genomes. To exploit this observation we constructed a novel mammalian-E. coli shuttle vector whose replication in E. coli is driven by the F sex factor episome origin. This new vector system should accept inserts well in excess of 100 kb, thus putting the cloning of mammalian genes by direct phenotypic complementation within reach.