Directed, efficient, and versatile modifications of the Drosophila genome by genomic engineering

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 φC31-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 φC31. Second, DNA integration by φC31 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.

[1]  M. Gonzalez-Gaitan,et al.  Spermatocyte cytokinesis requires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes , 2007, Development.

[2]  G M Rubin,et al.  Expression of baculovirus P35 prevents cell death in Drosophila. , 1994, Development.

[3]  Y. Jan,et al.  Two types of asymmetric divisions in the Drosophila sensory organ precursor cell lineage , 2000, Nature Cell Biology.

[4]  A. Suzuki,et al.  The PAR-aPKC system: lessons in polarity , 2006, Journal of Cell Science.

[5]  R. Nusse,et al.  Creating transgenic Drosophila by microinjecting the site-specific φC31 integrase mRNA and a transgene-containing donor plasmid , 2007, Nature Protocols.

[6]  K. Golic,et al.  Methods for homologous recombination in Drosophila. , 2008, Methods in molecular biology.

[7]  H. Bellen,et al.  Emerging technologies for gene manipulation in Drosophila melanogaster , 2005, Nature Reviews Genetics.

[8]  N. Perrimon,et al.  Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes , 2008, Nature Genetics.

[9]  F. Eisenhaber,et al.  Phosphorylation-Induced Autoinhibition Regulates the Cytoskeletal Protein Lethal (2) giant larvae , 2005, Current Biology.

[10]  C.-ting Wu,et al.  A Simple Polymerase Chain Reaction-Based Method for the Construction of Recombinase-Mediated Cassette Exchange Donor Vectors , 2008, Genetics.

[11]  W. Nelson,et al.  Adaptation of core mechanisms to generate cell polarity , 2003, Nature.

[12]  Hugo J. Bellen,et al.  P[acman]: A BAC Transgenic Platform for Targeted Insertion of Large DNA Fragments in D. melanogaster , 2006, Science.

[13]  C.-ting Wu,et al.  Site-Specific Transformation of Drosophila via ϕC31 Integrase-Mediated Cassette Exchange , 2006, Genetics.

[14]  Y. Rong,et al.  Gene targeting by homologous recombination in Drosophila. , 2000, Science.

[15]  B. Bainbridge,et al.  Genetics , 1981, Experientia.

[16]  Yuh Nung Jan,et al.  Adherens junctions inhibit asymmetric division in the Drosophila epithelium , 2001, Nature.

[17]  J. Gall,et al.  Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases , 2008, Proceedings of the National Academy of Sciences.

[18]  S. Tsukita,et al.  Real-time imaging of cell-cell adherens junctions reveals that Drosophila mesoderm invagination begins with two phases of apical constriction of cells. , 2001, Journal of cell science.

[19]  S. Tsukita,et al.  Nonchordate classic cadherins have a structurally and functionally unique domain that is absent from chordate classic cadherins. , 1999, Developmental biology.

[20]  B. Margolis,et al.  Apicobasal polarity complexes , 2005, Journal of Cell Science.

[21]  Toni Cathomen,et al.  Unexpected failure rates for modular assembly of engineered zinc fingers , 2008, Nature Methods.

[22]  Michele P Calos,et al.  Phage integrases: biology and applications. , 2004, Journal of molecular biology.

[23]  Michele P Calos,et al.  Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. , 2004, Genetics.

[24]  Y. Jan,et al.  Efficient Ends-Out Gene Targeting In Drosophila , 2008, Genetics.

[25]  G. Rubin,et al.  Targeted mutagenesis by homologous recombination in D. melanogaster. , 2002, Genes & development.

[26]  N. Perrimon,et al.  Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts , 2001, Nature.

[27]  Jeff J. Sekelsky,et al.  From sequence to phenotype: reverse genetics in drosophila melanogaster , 2002, Nature Reviews Genetics.

[28]  K. Golic,et al.  Ends-out, or replacement, gene targeting in Drosophila , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Jie Chen,et al.  A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in Drosophila , 2008, Proceedings of the National Academy of Sciences.

[30]  A. Wodarz.  Extraction and immunoblotting of proteins from embryos. , 2008, Methods in molecular biology.

[31]  R. Maeda,et al.  An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases , 2007, Proceedings of the National Academy of Sciences.

[32]  Eric C. Olivares,et al.  A phage integrase directs efficient site-specific integration in human cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.