Huang Juan, Zhou Wenke, Dong Wei, Watson Annie M, Hong Yang
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Proc Natl Acad Sci U S A. 2009 May 19;106(20):8284-9. doi: 10.1073/pnas.0900641106. Epub 2009 May 8.
With the completion of genome sequences of major model organisms, increasingly sophisticated genetic tools are necessary for investigating the complex and coordinated functions of genes. Here we describe a genetic manipulation system termed "genomic engineering" in Drosophila. Genomic engineering is a 2-step process that combines the ends-out (replacement) gene targeting with phage integrase phiC31-mediated DNA integration. First, through an improved and modified gene targeting method, a founder knock-out line is generated by deleting the target gene and replacing it with an integration site of phiC31. Second, DNA integration by phiC31 is used to reintroduce modified target-gene DNA into the native locus in the founder knock-out line. Genomic engineering permits directed and highly efficient modifications of a chosen genomic locus into virtually any desired mutant allele. We have successfully applied the genomic engineering scheme on 6 different genes and have generated at their loci more than 70 unique alleles.
随着主要模式生物基因组序列的完成，需要越来越复杂的遗传工具来研究基因的复杂和协调功能。在此，我们描述了一种在果蝇中称为“基因组工程”的遗传操作体系。基因组工程是一个两步过程，它将末端外显（置换）基因靶向与噬菌体整合酶phiC31介导的DNA整合相结合。首先，通过一种改进和改良的基因靶向方法，通过删除目标基因并用phiC31的整合位点进行替换来产生一个奠基性敲除品系。其次，利用phiC31介导的DNA整合将修饰后的目标基因DNA重新引入奠基性敲除品系的天然基因座中。基因组工程允许将选定的基因组位点定向且高效地修饰成几乎任何所需的突变等位基因。我们已成功地将基因组工程方案应用于6个不同的基因，并在它们的基因座上产生了70多个独特的等位基因。
