Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 106, Issue 20, Pages 8284-8289Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0900641106
Keywords
cell polarity; ends-out targeting; homologous recombination; phiC31 integrase
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Funding
- University of Pittsburgh Medical School
- National Center for Research Resources, National Institutes of Health [1R21RR024869]
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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 phi C31-mediated DNA integration. First, through an improved and modified gene targeting method, a founder knockout line is generated by deleting the target gene and replacing it with an integration site of phi C31. Second, DNA integration by phi C31 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.
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