A Novel Method for Floxed Gene Manipulation Using TAT-Cre Recombinase in Ex Vivo Precision-Cut Lung Slices (PCLS)

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. Key features • Achieve permanent ex vivo gene modifications in complex tissue-based models within four days. • Highly adaptable gene modification method that can be applied to induce gene deletion or activation. • Allows simple Cre dosage testing in a controlled ex vivo setting with the advantage of using PCLS generated from the same animal as true controls. • With optimisation, this method can be applied to precision-cut tissue slices of other organs.

Keywords: Ex vivo model; Floxed allele modification; Gene activation; Gene deletion; Gene modification; PCLS; Permanent gene manipulation; Precision-cut lung slices; TAT-Cre recombinase; TReATS.

Video 1.. Precision-cut lung slices (PCLS) generation using compresstome

Figure 1.. TReATS method activates the expression of EYFP transgene in precision-cut lung slices (PCLS) generated from R26R-EYFP mouse.

(A) Schematic showing a simplified structure of the loxP-modified allele in R26R-EYFP mouse and the structure of the targeted locus after Cre-mediated excision of the loxP-flanked stop sequence. TAT-Cre recombinase-mediated excision of the upstream loxP-flanked stop sequence allows the transcription and translation of EYFP transgene. (B) Tiled image shows ubiquitous expression of EYFP protein in TAT-Cre-treated R26R-EYFP PCLS. Image was generated using a confocal microscope.

Figure 2.. Live imaging of precision-cut lung slices (PCLS) stained with specific cell type markers.

(A) Schematic diagram shows the live imaging setup that utilises a transwell insert and a metal washer to secure the PCLS in place throughout the imaging process. (B-C) Representative images showing untreated R26R-EYFP PCLS (B) and TAT-Cre recombinase-treated R26R-EYFP PCLS (C). EYFP protein expression is shown in yellow and cell nuclei were labelled with DAPI (blue). Images were captured on a confocal microscope using an HC PL APO 10×/0.40 air objective lens. (D–H) Images showing co-localisation of EYFP with different alveolar cell type markers: LAMP3 (mature ATII cells) (D), PECAM for endothelial cells (E), PDPN for ATI cells (F), CD11c for macrophages (G), and vimentin for fibroblasts (H). Images were taken using 40× objective lens on a confocal microscope.

Figure 3.. Precision-cut lung slices (PCLS) homogenisation workflow