Fast and Efficient Drosophila melanogaster Gene Knock-Ins Using MiMIC Transposons

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][1] , and [sky][2] by using different MiMIC elements. We replaced 6 kb of genomic DNA encompassing the [tau][1] locus and 35 kb encompassing the [sky][2] 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 (<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. [1]: http://flybase.org/reports/FBgn0266579.html [2]: http://flybase.org/reports/FBgn0032901.html