Targeted genetics in Drosophila cell lines: Inserting single transgenes in vitro.
A long-standing problem with analyzing transgene expression in tissue-culture cells is the variation caused by random integration of different copy numbers of transfected transgenes. In mammalian cells, single transgenes can be inserted by homologous recombination but this process is inefficient in Drosophila cells. To tackle this problem, our group, and the Cherbas group, used recombination-mediated cassette exchange (RMCE) to introduce single-copy transgenes into specific locations in the Drosophila genome. In both cases, ϕC31 was used to catalyze recombination between its target sequences attP in the genome, and attB flanking the donor sequence. We generated cell lines de novo with a single attP-flanked cassette for recombination, whereas, Cherbas et al. introduced a single attP-flanked cassette into existing cell lines. In both approaches, a 2-drug selection scheme was used to select for cells with a single copy of the donor sequence inserted by RMCE and against cells with random integration of multiple copies. Here we describe the general advantages of using RMCE to introduce genes into fly cells, the different attributes of the 2 methods, and how future work could make use of other recombinases and CRISPR/Cas9 genome editing to further enable genetic manipulation of Drosophila cells in vitro.
a Department of Molecular and Human Genetics , Baylor College of Medicine , Houston TX.
Sathiya N
SN
Manivannan
b Department of Molecular Genetics , The Ohio State University , Columbus , OH.
Amanda
A
Simcox
D000818:Animals; D002460:Cell Line; D004330:Drosophila / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D017344:Genes, Insect; D066298:In Vitro Techniques; D045522:Recombinases / Q000378:metabolism; D011995:Recombination, Genetic; D019076:Transgenes
D045522:Recombinases
Drosophila; cell lines; recombination-mediated cassette exchange; transgene