Publication Date
8-9-2018
Journal
eLife
DOI
10.7554/eLife.38709
PMID
30091705
PMCID
6095692
Published Open-Access
yes
Keywords
Animals, Clustered Regularly Interspaced Short Palindromic Repeats, Drosophila, Entomology, Gene Targeting, Genes, Reporter, Integrases, Mutagenesis, Insertional
Abstract
We generated two new genetic tools to efficiently tag genes in Drosophila. The first, Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on Cre recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette. This sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.
Research organism: D. melanogaster
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