Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange.
Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ∼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied.
Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210 Molecular Cellular and Developmental Biology Program, Ohio State University, Columbus, Ohio 43210.
Sathiya N
SN
Manivannan
Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210.
Thomas L
TL
Jacobsen
Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210.
Peter
P
Lyon
Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210.
Bhavani
B
Selvaraj
Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210.
Peter
P
Halpin
Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210 Molecular Cellular and Developmental Biology Program, Ohio State University, Columbus, Ohio 43210 simcox.1@osu.edu.
Amanda
A
Simcox
D000818:Animals; D002460:Cell Line; D004331:Drosophila melanogaster / Q000235:genetics; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D017344:Genes, Insect; D019426:Integrases / Q000378:metabolism*; D008297:Male; D011995:Recombination, Genetic; D046509:Tissue Culture Techniques; D014162:Transfection / Q000379:methods*; D019076:Transgenes
D019426:Integrases
Drosophila; RMCE; cell line; single-transgene expression