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

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.

This protocol is used in: Dis.Model.Mech.(2023), DOI: 10.1242/dmm.050267Precision-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.

Background
Precision-cut lung slices (PCLS) represent a 3D ex vivo platform that plays a crucial role in advancing respiratory research.The significance of PCLS lies in their ability to serve as an intermediate model bridging conventional in vitro models and in vivo studies.PCLS preserve the spatial and cellular complexities of the native lung microenvironment while mitigating challenges associated with in vivo experiments, such as ethical considerations, time constraints, and cost limitations [1].As an ex vivo alternative, PCLS provide a controlled and physiologically relevant setting to investigate various aspects of lung biology, from responses to injury to the behaviour of specific cell types, all within the context of the native lung architecture [2][3][4][5].This advances our understanding of the nuanced interplay between cellular and molecular components governing respiratory function.Despite the versatility of PCLS, a notable gap has remained to enable effective and permanent gene manipulation in tissue slices, primarily due to the challenges associated with the inherent complexity of the tissue and the imperative to maintain tissue viability.Addressing this gap, our protocol describes a novel approach termed TReATS (TAT-Cre recombinase-mediated floxed allele modification in tissue slices).The TReATS method utilises TAT-Cre recombinase, a cell-permeant Cre protein, to effectively induce ex vivo genetic modifications in PCLS derived from floxed animals, resulting in permanent gene activation or deletion within a four-day timeframe [6].

Precision-cut lung slicing
a.In a 90 mm Petri dish, separate the fresh lung lobes using forceps and dissecting scissors.b.Slice a tiny section off from the basal end of the tissue to form a flat surface using a scalpel blade.This ensures that the tissue can be securely glued to the specimen tube.c.Squeeze a tiny drop of super glue onto the specimen tube base.
Note: Do not use too much super glue, as this will cause the specimen tube plunger to stick to the metal tube.d.Place the lung lobe vertically onto the glue using forceps and allow the super glue to cure for 1 min.e. Pull the specimen tube plunger downwards until the tissue is fully covered by the metal tube.f.Use a 5 mL syringe and pipette enough lukewarm agarose solution to cover the tissue and ensure no bubbles formed around it.g.Place the chilling block around the specimen tube for 1 min to solidify the agarose.h.Once the agarose is solidified (turns from transparent to translucent), insert the specimen tube into the buffer tray fully until the stopper ring touches the adapter.i. Adjust the micrometer until it touches the back of the specimen tube.

E. Live imaging of PCLS using confocal microscope
1. Pre-equilibrate the incubator chamber of an inverted confocal microscope with the following conditions: 37 °C, 5% CO 2 , and room air oxygen levels, approximately 21%, for 30 min.Note: It is crucial to equilibrate the incubator chamber before imaging to maintain the viability of PCLS throughout the imaging process, particularly if the imaging is expected to take over an hour.2. Meanwhile, add 100 μL of prewarmed imaging medium (see Recipes) into each well in an Ibidi uncoated 24-well µ-plate.Carefully transfer the immunostained PCLS from the 48-well plate onto the imaging medium using a metal spatula.3. Using forceps, gently place a 12 mm Millicell ® cell culture insert with 0.4 μm pores onto the PCLS so that the PCLS is positioned at the centre of the insert.Secure the insert in place by placing a flat metal washer on top of the insert (Figure 2A). 4. Add 200 μL and 400 μL of imaging media into the bottom chamber and upper chamber, respectively.Cover the 24-well plate with a lid. 5. Once the incubator chamber is equilibrated, place the Ibidi 24-well plate containing PCLS onto the motorised stage.Image using an HC PL APO 10×/0.40air objective lens (Figure 2B, 2C) or HC PL APO 40×/1.30oil objective lens (Figure 2D-2H).Use the following lasers: Diode 405 nm (for DAPI), argon 514 (for EYFP), Diode 561 (for vimentin), and Diode 633 (for LAMP3, PDPN, PECAM, and Cd11c) (Figure 2D-2H).6. Image five random fields of alveolar regions per PCLS at approximately 40 μm from the bottom of the PCLS.

Note: Low magnification (10× objective lens) is recommended for an overview of EYFP activation across the entire PCLS; high magnification (40× objective lens) is recommended to visualise co-localisation of EYFP with different alveolar cell type markers.
Note: A distance of 40 μm from the bottom of the PCLS is recommended as the PCLS are generally at the best focus at this distance.Imaging further from this distance is possible but can become challenging due to limited light penetrance through the depth of PCLS.To image the whole depth of PCLS, Z-stack is required, and this has been described in the original manuscript.It is also recommended to avoid imaging the PCLS less than 20 μm from the bottom of the PCLS, as the slicing process injures the surface of the PCLS, and an intact lung architecture may not be retained closer to the sliced surface.Thus, imaging at 40 μm from the bottom of the PCLS provides a convenient and representative snapshot of the PCLS.

F. Cell viability test (MTT assay)
1.At 72 h post-Cre treatment, perform MTT assays to examine whether the Cre treatment affects cell viability by assessing cell metabolic activities within PCLS.Note: Use PCLS of equal size generated from the middle of the lung lobe for MTT assays.b.Pipette the mixture up and down a few times to mix.c.Close the lids of the tubes and spin down to collect the solution to the bottom of the tubes.d.Load the samples onto the thermal cycler and start the run using the following protocol: 25 °C for 10 min, 37 °C for 2 h, 85 °C for 5 min, followed by indefinite holding at 4 °C.e. Store the cDNA samples at -20 °C freezer until use.f.After all the components are added to the tubes, close the caps and vortex briefly to mix.g.Briefly centrifuge the 8-tube PCR strips using a benchtop microfuge with 4 × 8 PCR strip rotor to spin down the contents.h.Pipette 10 μL of the qRT-PCR reaction mix into each well of a 96-well PCR plate.i. Seal the plate with an optical adhesive film.Centrifuge briefly using a microfuge with a 96-well plate rotor to eliminate the air bubbles.j.Run the PCR on a StepOne Plus Real-Time PCR System using the following parameters: UNG incubation at 50 °C for 2 min, polymerase activation at 95 °C for 20 s, followed by 40 cycles of (denaturation at 95 °C for 1 s and annealing at 60 °C for 20 s).k.Analyse the relative transcript levels using the 2 −ΔΔCT method (see Data analysis).
Note: Use four mice per genotype, pool three PCLS per RNA sample, and three RNA samples per treatment per experiment.

1 .
Preparation a. Cool the chilling block in the -20 °C freezer for 1 h before use.b.Attach the stainless-steel blade to the blade holder using super glue.Use a paper clip to hold it in place and air dry at room temperature for 10 min before use.c.Prepare 200 mL of HBSS/HEPES buffer (see Recipes) and a 48-well plate with ice-cold SF-DMEM (300 μL/well) and keep on ice until use.d.Disinfect the surgical tools, spatula, specimen holder, and the buffer tray of the compresstome by wiping them with 70% ethanol (see Recipes).Let them air dry.e. Prepare 50 mL of 2% (w/v) agarose solution (see Recipes) and an icebox with lukewarm water to keep the agarose warm and prevent it from solidifying throughout the slicing process.
j. Slide the blade holder into the axial bar of the vibrating unit and secure it with the Allen key.k.Add ice-cold HBSS/HEPES buffer into the buffer tray until the specimen tube is submerged in the buffer.l.Set the compresstome to start slicing at a thickness of 300 μm, advance speed 4, and oscillation 4. m.The desired thickness is 250 μm.Start slicing on continuous mode at 300 μm and reduce the thickness by 10 μm after each slice is produced (300 μm → 290 μm → 280 μm → 270 μm → 260 μm → 250 μm) until reaching 250 μm.Continue slicing at the thickness of 250 μm until the entire embedded tissue is completely sliced.Note: The gradual reduction in thickness helps to produce slices with consistent and accurate thickness.n.Collect the PCLS with a metal spatula and place them into the 48-well plate containing 300 μL of SF-DMEM (one PCLS per well) as the slicing progresses.Note: As the number of live cells is a critical factor that determines the metabolic activity in MTT assays, it is recommended to use PCLS generated from the middle of the lung lobe to ensure size conformity.o.Incubate the PCLS in the incubator (37 °C, 5% CO2) for 2 h before proceeding with the washing steps.p.After incubation, wash the PCLS with 300 μL of prewarmed SF-DMEM per well.Incubate at 37 °C in 5% CO2 for 5 min during each wash and repeat the washing step three times.Pause point: The PCLS can be incubated overnight at 37 °C in 5% CO2 before proceeding with the washing steps the next day.This does not significantly affect the efficacy of TAT-Cre treatment.C. TAT-Cre treatment of PCLS 1. Experimental controls: a. Negative control for Cre protein Treat PCLS from the same R26R-EYFP mouse with only SF-DMEM.These PCLS serve as the negative control to exclude leaky transgene.b.Control for artefacts due to the TAT-Cre treatment Use PCLS from a wildtype mouse of the same background strain as the transgenic mouse as a control to exclude artefacts caused by the TAT-Cre treatment.2. TAT-Cre recombinase treatment a.After the washing steps, add 3 μM of TAT-Cre recombinase solution diluted in prewarmed SF-DMEM to the R26R-EYFP and wildtype mouse PCLS (250 μL/well) and incubate for 24 h at 37 °C in 5% CO2 (Figure 1A).Untreated R26R-EYFP PCLS with SF-DMEM are used as negative controls.b.After 24 h of incubation, remove the TAT-Cre solution.Add fresh prewarmed SF-DMEM into the PCLS (250 μL/well) and incubate at 37 °C in 5% CO2 for a further 48 h.See Troubleshooting.

Figure 1 . 9 Published: Apr 20, 2024 D. Live staining of PCLS 1 . 2 .
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 loxPmodified 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 .
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×/0.40air objective lens.(D-H) Images showing colocalisation 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.

2 .G. Validation of gene expression change 1 .
Prepare 10% MTT solution (see Recipes).Use 250 μL of 10% MTT solution for each PCLS per well in a 48-well plate.3. Positive control for dead cells: PCLS treated with 70% methanol serve as control for dead cells.Add 250 μL of 70% methanol (see Recipes) into the control PCLS and incubate at room temperature for 15 min.After 15 min, remove the methanol solution and wash the PCLS three times with 300 μL of room-Published: Apr 20, 2024 temperature PBS.Proceed with step F4. 4. Remove the culture medium.Add 250 μL of 10% MTT solution into the PCLS and incubate at 37 °C and 5% CO2 in the dark for 45 min.5.After incubation, discard the MTT solution.Add 250 μL of DMSO into each PCLS and incubate at 37 °C and 5% CO2 in the dark for a further 10 min.6.After DMSO incubation, gently pipette up and down a few times and transfer 200 μL from each well into a new clear-bottom 96-well plate.7. Read the absorbance at 570 nm and 690 nm and calculate cell viability (see Data analysis).Note: Use four mice per genotype and three PCLS per treatment per experiment.PCLS homogenisation and RNA extraction a. Use RNase decontamination solution to clean the working surface.b.Steps G1b-g are illustrated in Figure 3.In each homogenisation tube, add homogenisation beads to the level of the bottom cone line of the tube.Label each tube with the sample name.

Published: Apr 20, 2024 4 .
Quantitative real-time PCR (qRT-PCR) a. Thaw cDNA samples on ice and measure the concentration of the cDNA samples using a Nanodrop.Use nuclease-free water as blank.b.Dilute the cDNA samples to 50 ng/μL using nuclease-free water.c.Thaw TaqMan ® assays on ice.Note: Use appropriate target assays accordingly.YFP (Assay ID: Mr04097229_mr) and B2m (Assay ID: Mm00437762_m1; reference gene) assays are used in this protocol (see Reagents).d.Mix the Fast Advanced Master Mix by pipetting up and down a few times.e. Calculate and prepare the qRT-PCR reaction mix in 8-tube PCR strips according to

Table 2 (
volume per reaction × number of reactions): Prepare three reactions (triplicates) per gene for each cDNA sample.