Fast and efficient Drosophila melanogaster gene knock-ins using MiMIC transposons.

Sven Vilain, Roeland Vanhauwaert, Ine Maes, Nils Schoovaerts, Lujia Zhou, Sandra Soukup, Raquel da Cunha, Elsa Lauwers, Mark Fiers, Patrik Verstreken
G3. 2014-10-08; 4(12): 2381-2387
DOI: 10.1534/g3.114.014803

PubMed
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1. G3 (Bethesda). 2014 Oct 8;4(12):2381-7. doi: 10.1534/g3.114.014803.

Fast and efficient Drosophila melanogaster gene knock-ins using MiMIC
transposons.

Vilain S(1), Vanhauwaert R(1), Maes I(1), Schoovaerts N(1), Zhou L(1), Soukup
S(1), da Cunha R(1), Lauwers E(1), Fiers M(2), Verstreken P(3).

Author information:
(1)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.
(2)VIB Center for the Biology of Disease, 3000 Leuven, Belgium.
(3)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 .

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 (


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