期刊
G3-GENES GENOMES GENETICS
卷 4, 期 12, 页码 2381-2387出版社
GENETICS SOCIETY AMERICA
DOI: 10.1534/g3.114.014803
关键词
Drosophila; genome editing homologous recombination; MiMIC
资金
- Fonds Wetenschappelijk Onderzoek (FWO) fellowship [81207]
- Flanders innovation agency (IWT) fellowship [121324, 111352]
- Fundacao para a Ciencia e a Tecnologia (FCT) fellowship [SFRH/BD/70027/2010]
- Research fund KU Leuven (KUL) [GOA/13/017]
- CREA [ZKC6335]
- European Research Council Starting Grant (ERC StG) [260678]
- Fonds Wetenschappelijk Onderzoek (FWO) [G094011N, G095511N, G053913N, G079013N]
- Hercules Foundation [AKUL/09/37, AKUL/11/30]
- Interuniversitaire Attractiepolen- Belgian Science Policy Office (IUAP-BELSPO) [P7/16]
- Vlaams Instituut voor Biotechnologie (VIB)
- Fundação para a Ciência e a Tecnologia [SFRH/BD/70027/2010] Funding Source: FCT
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 LRRKHA knock in allele. Given that similar to 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据