DNA oligonucleotide-assisted genetic manipulation increases transformation and homologous recombination efficiencies: Evidence from gene targeting of Dictyostelium discoideum

Artificial gene alteration by homologous recombination in living cells, termed gene targeting, presents fundamental and considerable knowledgeof in vivo gene function. In principle, this method can possibly be applied to any type of genes and transformable cells. However, its success islimited due to a low frequency of homologous recombination between endogenous targeted gene and exogenous transgene. Here, we describea general gene-targeting method in which co-transformation of DNA oligonucleotides (oligomers) could significantly increase the homologousrecombination frequency and transformation efficiency. The oligomers were simply designed such that they were identical to both the ends of thehomologous flanking regions of the targeting construct. Using this strategy, both targeted alleles of diploid cells were simultaneously replaced in asingle transformation procedure. Thus, the simplicity and versatility of this method applicable to any type of cell may increase the application ofgene targeting