Journal
NUCLEIC ACIDS RESEARCH
Volume 50, Issue 15, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkac448
Keywords
-
Categories
Funding
- NIDDK [ZIA DK075111]
- Division of Intramural Research (DIR), National Institute of Diabetes and Digestive and Kidney Diseases
Ask authors/readers for more resources
The combination of genome-editing and epitope tagging provides a powerful strategy to study proteins while preserving their physiological expression patterns. A simple and fast strategy to generate stable, endogenously tagged alleles is presented, allowing for the emulation of wild type protein expression and functionality.
The combination of genome-editing and epitope tagging provides a powerful strategy to study proteins with high affinity and specificity while preserving their physiological expression patterns. However, stably modifying endogenous genes in cells that do not allow for clonal selection has been challenging. Here, we present a simple and fast strategy to generate stable, endogenously tagged alleles in a non-transformed cell culture model. At the example of piwi in Drosophila ovarian somatic sheath cells, we show that this strategy enables the generation of an N-terminally tagged protein that emulates the expression level and subcellular localization of the wild type protein and forms functional Piwi-piRNA complexes. We further present a concise workflow to establish endogenously N-terminally and C-terminally tagged proteins, and knockout alleles through rapid selection of cell pools in fly and human models.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available