Fast and efficient drosophila melanogaster gene knock-ins using MiMIC transposons

VILAIN S. P. L., Vanhauwaert R., Maes I., Schoovaerts N., Soukup L. Z. S., da Cunha R., ...More

G3: Genes, Genomes, Genetics, vol.4, no.12, pp.2381-2387, 2014 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 4 Issue: 12
  • Publication Date: 2014
  • Doi Number: 10.1534/g3.114.014803
  • Journal Name: G3: Genes, Genomes, Genetics
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.2381-2387
  • Keywords: Drosophila, Genome editing, Homologous, MiMIc, Recombination
  • Istanbul Medipol University Affiliated: Yes


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 break2mediated 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 6kb of genomic DNA encompassing the tau locus and 35kb encompassing the sky locus with a recombination cassette that permits easy integration of DNA at these loci and we also generated a functional LRRKHA knock in allele. Given that 92% of the Drosophila genes are located within the vicinity (<35kb) 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.