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 knowledge of in vivo gene function. In principle, this method can possibly be applied to any type of genes and transformable cells. However, its success is limited due to a low frequency of homologous recombination between endogenous targeted gene and exogenous transgene. Here, we describe a general gene-targeting method in which co-transformation of DNA oligonucleotides (oligomers) could significantly increase the homologous recombination frequency and transformation efficiency. The oligomers were simply designed such that they were identical to both the ends of the homologous flanking regions of the targeting construct. Using this strategy, both targeted alleles of diploid cells were simultaneously replaced in a single transformation procedure. Thus, the simplicity and versatility of this method applicable to any type of cell may increase the application of gene targeting.
Kuwayama
Hidekazu
H
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Tennodai 1-1-1, Ibaraki 305-8572, Japan. hidekuwayama@biol.tsukuba.ac.jp
Yanagida
Toshio
T
Ueda
Masahiro
M
D000818:Animals; D016054:DNA, Protozoan / Q000235:genetics; D004023:Dictyostelium / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods; D008957:Models, Genetic; D009841:Oligonucleotides / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics*
D016054:DNA, Protozoan; D009841:Oligonucleotides