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

详细信息    查看全文

• 作者：Hidekazu Kuwayama ; Toshio Yanagida ; Masahiro Ueda
• 关键词：Dictyostelium discoideum ; Homologous recombination ; Gene targeting ; Transformation
• 刊名：Journal of Biotechnology
• 出版年：2008
• 出版时间：29 February 2008
• 年：2008
• 卷：133
• 期：4
• 页码：418-423
• 全文大小：514 K

文摘

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.