title: Fast and efficient Drosophila melanogaster gene knock-ins using MiMIC transposons.
abstract: Modern molecular genetics studies necessitate the manipulation of genes in their endogenous locus, but most of the current methodologies require an inefficient donor-dependent homologous recombination step to locally modify the genome. Here we describe a methodology to efficiently generate Drosophila knock-in alleles by capitalizing on the availability of numerous genomic MiMIC transposon insertions carrying recombinogenic attP sites. Our methodology entails the efficient PhiC31-mediated integration of a recombination cassette flanked by unique I-SceI and/or I-CreI restriction enzyme sites into an attP-site. These restriction enzyme sites allow for double-strand break-mediated removal of unwanted flanking transposon sequences, while leaving the desired genomic modifications or recombination cassettes. As a proof-of-principle, we mutated LRRK, tau, and sky by using different MiMIC elements. We replaced 6 kb of genomic DNA encompassing the tau locus and 35 kb encompassing the sky locus with a recombination cassette that permits easy integration of DNA at these loci and we also generated a functional LRRK(HA) knock in allele. Given that ~92% of the Drosophila genes are located within the vicinity (<35 kb) of a MiMIC element, our methodology enables the efficient manipulation of nearly every locus in the fruit fly genome without the need for inefficient donor-dependent homologous recombination events.
authors.affiliation: Department of Human Genetics and Leuven Research Institute for Neuroscience and Disease (LIND), KU Leuven, 3000 Leuven, Belgium VIB Center for the Biology of Disease, 3000 Leuven, Belgium.
authors.forename: Sven
authors.initials: S
authors.lastname: Vilain
authors.affiliation: Department of Human Genetics and Leuven Research Institute for Neuroscience and Disease (LIND), KU Leuven, 3000 Leuven, Belgium VIB Center for the Biology of Disease, 3000 Leuven, Belgium.
authors.forename: Roeland
authors.initials: R
authors.lastname: Vanhauwaert
authors.affiliation: Department of Human Genetics and Leuven Research Institute for Neuroscience and Disease (LIND), KU Leuven, 3000 Leuven, Belgium VIB Center for the Biology of Disease, 3000 Leuven, Belgium.
authors.forename: Ine
authors.initials: I
authors.lastname: Maes
authors.affiliation: Department of Human Genetics and Leuven Research Institute for Neuroscience and Disease (LIND), KU Leuven, 3000 Leuven, Belgium VIB Center for the Biology of Disease, 3000 Leuven, Belgium.
authors.forename: Nils
authors.initials: N
authors.lastname: Schoovaerts
authors.affiliation: Department of Human Genetics and Leuven Research Institute for Neuroscience and Disease (LIND), KU Leuven, 3000 Leuven, Belgium VIB Center for the Biology of Disease, 3000 Leuven, Belgium.
authors.forename: Lujia
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Department of Human Genetics and Leuven Research Institute for Neuroscience and Disease (LIND), KU Leuven, 3000 Leuven, Belgium VIB Center for the Biology of Disease, 3000 Leuven, Belgium.
authors.forename: Sandra
authors.initials: S
authors.lastname: Soukup
authors.affiliation: Department of Human Genetics and Leuven Research Institute for Neuroscience and Disease (LIND), KU Leuven, 3000 Leuven, Belgium VIB Center for the Biology of Disease, 3000 Leuven, Belgium.
authors.forename: Raquel
authors.initials: R
authors.lastname: da Cunha
authors.affiliation: Department of Human Genetics and Leuven Research Institute for Neuroscience and Disease (LIND), KU Leuven, 3000 Leuven, Belgium VIB Center for the Biology of Disease, 3000 Leuven, Belgium.
authors.forename: Elsa
authors.initials: E
authors.lastname: Lauwers
authors.affiliation: VIB Center for the Biology of Disease, 3000 Leuven, Belgium.
authors.forename: Mark
authors.initials: M
authors.lastname: Fiers
authors.affiliation: Department of Human Genetics and Leuven Research Institute for Neuroscience and Disease (LIND), KU Leuven, 3000 Leuven, Belgium VIB Center for the Biology of Disease, 3000 Leuven, Belgium patrik.verstreken@med.kuleuven.
authors.forename: Patrik
authors.initials: P
authors.lastname: Verstreken
mesh_terms: D000483:Alleles; D000818:Animals; D004251:DNA Transposable Elements / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics*; D055879:Gene Knock-In Techniques; D059765:Homologous Recombination; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D016875:tau Proteins / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; D029721:Drosophila Proteins; D016875:tau Proteins; C543697:LRRK protein, Drosophila; D017346:Protein Serine-Threonine Kinases
keywords: Drosophila; MiMIC; genome editing; homologous recombination