Originally published as Genetics Published Articles Ahead of Print on March 17, 2006.

Genetics, Vol. 173, 769-777, June 2006, Copyright © 2006
doi:10.1534/genetics.106.056945

Site-Specific Transformation of Drosophila via {phi}C31 Integrase-Mediated Cassette Exchange

* Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 and {dagger} Division of Genetics and Division of Molecular Medicine, Harvard Medical School, Boston, Massachusetts 02115

2 Corresponding author: Harvard Medical School, Department of Genetics, 77 Avenue Louis Pasteur, New Research Building, Room 264, Boston, MA 02115.
E-mail: twu{at}genetics.med.harvard.edu

Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the {phi}C31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, {phi}C31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs.




This article has been cited by other articles:


Home page
CSH ProtocolsHome page
G. Gao, N. Wesolowska, and Y. S. Rong
SIRT Combines Homologous Recombination, Site-Specific Integration, and Bacterial Recombineering for Targeted Mutagenesis in Drosophila
CSH Protocols, June 1, 2009; 2009(6): pdb.prot5236 - pdb.prot5236.
[Abstract] [Full Text]


Home page
Proc. Natl. Acad. Sci. USAHome page
J. Huang, W. Zhou, W. Dong, A. M. Watson, and Y. Hong
From the Cover: Directed, efficient, and versatile modifications of the Drosophila genome by genomic engineering
PNAS, May 19, 2009; 106(20): 8284 - 8289.
[Abstract] [Full Text] [PDF]


Home page
ScienceHome page
C. M. Choi, S. Vilain, M. Langen, S. Van Kelst, N. De Geest, J. Yan, P. Verstreken, and B. A. Hassan
Conditional Mutagenesis in Drosophila
Science, April 3, 2009; 324(5923): 54 - 54.
[Abstract] [Full Text] [PDF]


Home page
DevelopmentHome page
H. Jia, Y. Liu, W. Yan, and J. Jia
PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation
Development, January 15, 2009; 136(2): 307 - 316.
[Abstract] [Full Text] [PDF]


Home page
GeneticsHome page
J. R. Bateman and C.-t. Wu
A Simple Polymerase Chain Reaction-Based Method for the Construction of Recombinase-Mediated Cassette Exchange Donor Vectors
Genetics, November 1, 2008; 180(3): 1763 - 1766.
[Abstract] [Full Text] [PDF]


Home page
Stem CellsHome page
B. Thyagarajan, Y. Liu, S. Shin, U. Lakshmipathy, K. Scheyhing, H. Xue, C. Ellerstrom, R. Strehl, J. Hyllner, M. S. Rao, et al.
Creation of Engineered Human Embryonic Stem Cell Lines Using phiC31 Integrase
Stem Cells, January 1, 2008; 26(1): 119 - 126.
[Abstract] [Full Text] [PDF]


Home page
DevelopmentHome page
K. J. T. Venken and H. J. Bellen
Transgenesis upgrades for Drosophila melanogaster
Development, October 15, 2007; 134(20): 3571 - 3584.
[Abstract] [Full Text] [PDF]


Home page
Nucleic Acids ResHome page
M. Gupta, R. Till, and M. C. M. Smith
Sequences in attB that affect the ability of {phi}C31 integrase to synapse and to activate DNA cleavage
Nucleic Acids Res., May 11, 2007; 35(10): 3407 - 3419.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
J. Bischof, R. K. Maeda, M. Hediger, F. Karch, and K. Basler
An optimized transgenesis system for Drosophila using germ-line-specific {varphi}C31 integrases
PNAS, February 27, 2007; 104(9): 3312 - 3317.
[Abstract] [Full Text] [PDF]