A novel method for floxed gene manipulation using TAT-Cre recombinase in ex vivo precision-cut lung slices (PCLS)
File(s)
Published version
Author(s)
Cheong, Sek Shir
Luis, Tiago
Hind, Matthew
Dean, Charlotte
Type
Journal Article
Abstract
Precision-cut lung slices (PCLS), ex vivo 3D lung tissue models, have been widely used for various applications in lung research. PCLS serve as an excellent intermediary between in vitro and in vivo models because they retain all resident cell types within their natural niche while preserving the extracellular matrix environment. This protocol describes the TReATS (TAT-Cre recombinase-mediated floxed allele modification in tissue slices) method that enables rapid and efficient gene modification in PCLS derived from adult floxed animals. Here, we present detailed protocols for the TReATS method, consisting of two simple steps: PCLS generation and incubation in a TAT-Cre recombinase solution. Subsequent validation of gene modification involves live staining and imaging of PCLS, quantitative real-time PCR, and cell viability assessment. This four-day protocol eliminates the need for complex Cre-breeding, circumvents issues with premature lethality related to gene mutation, and significantly reduces the use of animals. The TReATS method offers a simple and reproducible solution for gene modification in complex ex vivo tissue-based models, accelerating the study of gene function, disease mechanisms, and the discovery of drug targets.
Date Issued
2024-04-20
Date Acceptance
2024-03-19
Citation
Bio-protocol, 2024, 14 (8)
ISSN
2331-8325
Publisher
Bio-Protocol
Journal / Book Title
Bio-protocol
Volume
14
Issue
8
Copyright Statement
© 2024 The Authors; exclusive licensee Bio-protocol LLC.
This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/)
This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/)
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Publication Status
Published