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One-step generation of auxin-inducible degron cells with high-efficiency homozygous tagging
One-step generation of auxin-inducible degron cells with high-efficiency homozygous tagging
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Length:
20 minutes
Released:
Mar 27, 2023
Format:
Podcast episode
Description
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2023.03.26.534268v1?rss=1
Authors: Li, S., Wang, Y., Stoel, M. v. d., Zhou, X., Madhusudan, S., Kanerva, K., Nguyen, V. D., Eskici, N., Olkkonen, V. M., Zhou, Y., Raivio, T., Ikonen, E.
Abstract:
Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with greater than 80% average homozygous degron-tagging efficiency in A431 cells, and greater than 50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells.
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http://biorxiv.org/cgi/content/short/2023.03.26.534268v1?rss=1
Authors: Li, S., Wang, Y., Stoel, M. v. d., Zhou, X., Madhusudan, S., Kanerva, K., Nguyen, V. D., Eskici, N., Olkkonen, V. M., Zhou, Y., Raivio, T., Ikonen, E.
Abstract:
Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with greater than 80% average homozygous degron-tagging efficiency in A431 cells, and greater than 50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells.
Copy rights belong to original authors. Visit the link for more info
Podcast created by Paper Player, LLC
Released:
Mar 27, 2023
Format:
Podcast episode
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