In this proposal we describe the development of a novel genetic manipulation system termed """"""""genomic engineering"""""""" that will permit efficient, unlimited and directed modification of a chosen genomic locus in Drosophila. Genomic engineering is a two-step process. First, through an optimized gene targeting routine, a founder knock-out line is generated by deleting the target gene and replacing it with a small DNA recombination site of phage integrase FC31. Second, DNA integration by FC31 is used to reintroduce modified target gene DNA into the native locus in the founder knock-out line. Since FC31 mediated DNA integration is highly efficient and non-discriminating, virtually any desired genetic modification can be generated with high throughput efficiency. Genomic engineering will have a profound and revolutionary impact as a tool for directed engineering of the Drosophila genome. First, genomic engineering overcomes the inherent inefficiency and limitations of homologous recombination. It provides a virtually unlimited approach to generate any desired mutant allele of a target gene for genetic, biochemical and cell biologic assays. Second, for constructing useful and informative human disease models, genomic engineering makes it possible to precisely replicate the human genetic lesions into the Drosophila homologous genes. Finally, by systematically generating founder knock-out lines for conserved and essential genes in the long term, we will transform Drosophila into a far more efficient and versatile genetic model organism that will also be vastly more accessible and attractive to non-fly researchers for addressing their biological questions.

Public Health Relevance

Drosophila is a leading genetic model system for addressing crucial biological questions in human diseases. Genomic engineering is a new powerful genetic tool that will significantly facilitate the analysis of complex disease pathways and the construction of better disease models in Drosophila.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21RR024869-02
Application #
7746473
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
O'Neill, Raymond R
Project Start
2008-12-12
Project End
2010-11-30
Budget Start
2009-12-01
Budget End
2010-11-30
Support Year
2
Fiscal Year
2010
Total Cost
$170,438
Indirect Cost
Name
University of Pittsburgh
Department
Physiology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Chen, Yi-Jiun; Huang, Juan; Huang, Lynn et al. (2017) Phosphorylation potential of Drosophila E-Cadherin intracellular domain is essential for development and adherens junction biosynthetic dynamics regulation. Development 144:1242-1248
Dong, Wei; Zhang, Xuejing; Liu, Weijie et al. (2015) A conserved polybasic domain mediates plasma membrane targeting of Lgl and its regulation by hypoxia. J Cell Biol 211:273-86
Zhou, Wenke; Hong, Yang (2012) Drosophila Patj plays a supporting role in apical-basal polarity but is essential for viability. Development 139:2891-6
Zhou, Wenke; Huang, Juan; Watson, Annie M et al. (2012) W::Neo: a novel dual-selection marker for high efficiency gene targeting in Drosophila. PLoS One 7:e31997
Huang, Juan; Ghosh, Pallavi; Hatfull, Graham F et al. (2011) Successive and targeted DNA integrations in the Drosophila genome by Bxb1 and phiC31 integrases. Genetics 189:391-5
Huang, Juan; Huang, Lynn; Chen, Yi-Jiun et al. (2011) Differential regulation of adherens junction dynamics during apical-basal polarization. J Cell Sci 124:4001-13
Huang, Juan; Zhou, Wenke; Dong, Wei et al. (2009) From the Cover: Directed, efficient, and versatile modifications of the Drosophila genome by genomic engineering. Proc Natl Acad Sci U S A 106:8284-9