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Elife. 2018 Aug 9;7. pii: e38709. doi: 10.7554/eLife.38709.

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila.

Author information

1
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
2
Department of Biochemistry and Cell Biology, Rice University Houston, Houston, United States.
3
Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States.
4
Program in Developmental Biology, Baylor College of Medicine, Houston, United States.
5
Department of Neuroscience, Baylor College of Medicine, Houston, United States.
6
Jan and Dan Duncan Neurological Research Institute, Houston, United States.
#
Contributed equally

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.

KEYWORDS:

D. melanogaster; Double Header; T2A-GAL4; Trojan exons; gene editing; genetics; genomics; protein tagging; swappable insertion cassette

PMID:
30091705
PMCID:
PMC6095692
DOI:
10.7554/eLife.38709
[Indexed for MEDLINE]
Free PMC Article

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