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

使用TAT-Cre重组酶在体外精确切片的肺组织中操作Floxed基因的新方法

SC Sek-Shir Cheong
TL Tiago C. Luis
MH Matthew Hind
CD Charlotte H. Dean

发布: 2024年04月20日第14卷第8期 DOI: 10.21769/BioProtoc.4980 浏览次数: 2137

评审: Alessandro DidonnaIvan ShapovalovAnonymous reviewer(s)

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Oct 2023

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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: Precision-cut lung slices (精确切片的肺组织) search PCLS (PCLS) search TAT-Cre recombinase (TAT-Cre重组酶) search Gene modification (基因修改) search Floxed allele modification (Floxed等位基因修改) search Ex vivo model (体外模型) search TReATS (TReATS) search Permanent gene manipulation (永久性基因操作) search Gene deletion (基因删除) search Gene activation (基因激活) search

Graphical overview


Workflow of TAT-Cre recombinase-mediated floxed allele modification in tissue slices (TReATS) and validation protocols of gene modification

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 [2–5]. 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]. This simple and effective technique circumvents the need for a Cre-containing mouse allele, eliminating the necessity for complex breeding strategies to generate transgenic mice of interest, thereby remarkedly reducing the time and costs involved and, most importantly, aligning with the 3Rs principles (Refinement, Reduction, and Replacement) by minimising the use of animals in research.

Application of the TReATS method in adult PCLS has important implications for genetic studies, as it overcomes issues related to embryonic or perinatal lethality that can occur upon early gene ablation or overexpression, thereby enabling the possibility to investigate gene function in adulthood [6]. While the TReATS method is useful for inducing ubiquitous pan-tissue gene manipulation across PCLS, its current limitation lies in its inability to induce cell type–specific gene modification. This can only be achieved by crossing with animals possessing specific Cre drivers.

The TReATS method is versatile and adaptable, as evidenced in the original manuscript, where the method is successfully applied to different loxP-modified alleles, inducing gene deletion or activation [6]. This adaptability holds great promise for accelerating the discovery of gene function, unravelling disease mechanisms, and exploring potential therapeutic interventions.

Materials and reagents

Biological materials

  1. Rosa26-flox-stop-flox-EYFP mouse (10–12 weeks; male/female) (from Dr. Tiago Luis; strain: 006148; RRID: IMSR_JAX:006148). Abbreviation: R26R-EYFP

  2. Wildtype C57BL/6J mouse (10–12 weeks; male/female; serves as a negative control) (Charles River, catalog number: 632)


Reagents

  1. Dulbecco’s modified Eagle medium (DMEM-Glutamax) (Sigma-Aldrich, catalog number: 31966021)

  2. Phenol red–free DMEM (Life Technologies, catalog number: 21063029)

  3. Low-gelling temperature agarose (Sigma-Aldrich, catalog number: A9414)

  4. Hank's balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H6648)

  5. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (Life Technologies, catalog number: 15630080)

  6. Penicillin-streptomycin 10,000 U/mL (Merck Life Science UK, catalog number: P0781)

  7. Phosphate buffered saline (PBS) (Life Technologies, catalog number: 10010056)

  8. Super glue liquid precision bottle 5 g (Loctite, catalog number: 2632836)

  9. TAT-Cre recombinase (Merck, catalog number: SCR508)

  10. ProlongTM live antifade reagent (Invitrogen, catalog number: P36975)

  11. Cell proliferation kit (MTT assay) (Roche, catalog number: 11465007001)

  12. Dimethyl sulfoxide (DMSO) (Merck Life Science UK, catalog number: D5879)

  13. Absolute ethanol for molecular biology (Sigma-Aldrich, catalog number: 51976)

  14. Methanol (Sigma-Aldrich, catalog number: 34860)

  15. RNase Zap RNase decontamination solution (Life Technologies, catalog number: AM9780)

  16. Ultrapure DNase/RNase-free distilled water (Life Technologies, catalog number: 10977035)

  17. RNeasy mini kit (Qiagen, catalog number: 74104)

  18. RNase-free DNase set (Qiagen, catalog number: 79254)

  19. RNA screen tape (Agilent, catalog number: 5067-5576)

  20. RNA screen tape sample buffer (Agilent, catalog number: 5067-5577)

  21. High-capacity cDNA reverse transcription kit (Applied Biosystems, catalog number: 4368814)

  22. TaqMan fast advanced master mix (Life Technologies, catalog number: 4444556)

  23. (Optional) Mouse B2m gene expression assay (Life Technologies, catalog number: 4331182_Mm00437762_m1)

  24. (Optional) YFP gene expression assay (Life Technologies, catalog number: 4331182_ Mr04097229_mr)


Solutions

  1. HBSS/HEPES buffer (see Recipes)

  2. Agarose solution 2% (w/v) (see Recipes)

  3. Serum-free (SF)-DMEM culture medium (see Recipes)

  4. Imaging medium (see Recipes)

  5. MTT working solution (see Recipes)

  6. 70% (v/v) methanol (see Recipes)

  7. 70% (v/v) ethanol (see Recipes)

  8. RLT/β-mercaptoethanol lysis buffer (see Recipes)


Recipes

  1. HBSS/HEPES buffer (store at 4 °C for up to six months)

    ReagentFinal concentrationVolume
    HBSSn/a495 mL
    HEPES10 mM5 mL
    Totaln/a500 mL


  2. Agarose solution 2% (w/v) (freshly prepared and used)

    ReagentFinal concentrationQuantity
    HBSS/HEPESn/a50 mL
    Agarose2% (w/v)1 g
    Totaln/a50 mL


  3. SF-DMEM culture medium (store at 4 °C for up to six months)

    ReagentFinal concentrationVolume
    DMEM-Glutamaxn/a495 mL
    Penicillin-streptomycin100 U/mL5 mL
    Totaln/a500 mL


  4. Imaging medium (freshly prepared and used)

    ReagentFinal concentrationVolume
    Phenol red–free DMEMn/a49 mL
    Penicillin-streptomycin100 U/mL500 µL
    ProlongTM live antifade reagent1% (v/v)500 µL
    Totaln/a50 mL


  5. MTT working solution (freshly prepared and used)

    ReagentFinal concentrationVolume
    SF-DMEMn/a2.7 mL
    MTT solution (Cell proliferation kit)10% (v/v)300 µL
    Totaln/a3 mL


  6. 70% methanol (store at room temperature)

    ReagentFinal concentrationVolume
    Methanol70% (v/v)14 mL
    H2On/a6 mL
    Totaln/a20 mL


  7. 70% ethanol (store at room temperature)

    ReagentFinal concentrationVolume
    Absolute ethanol70% (v/v)14 mL
    H2On/a6 mL
    Totaln/a20 mL


  8. RLT/β-mercaptoethanol lysis buffer (freshly prepared and used)

    ReagentFinal concentrationAmount
    RLT buffer (RNeasy mini kit)n/a1,980 µL
    β-mercaptoethanol1% (v/v)20 µL
    Totaln/a2 mL


Antibodies and fluorescent dyes

  1. Alexa Fluor® 647-conjugated rat anti-lysosomal associated membrane protein 3 (LAMP3) (marker for mature differentiated alveolar type II (ATII) cells) (Dendritics, catalog number: DDX0192A647), dilution: 1:250

  2. Podoplanin eFluor® 660 [alveolar type I (ATI) cell marker] (Life Technologies, catalog number: 50-5381-82), dilution: 1:500

  3. Alexa Fluor® 647-conjugated platelet endothelial cell adhesion molecule (PECAM) (vascular endothelium marker) (BioLegend, catalog number: 102416), dilution: 1:200

  4. Alexa Fluor® 647-conjugated CD11c (macrophage marker) (BioLegend, catalog number: 117312), dilution: 1:200

  5. BioTrackerTM TiY vimentin live cell dye (fibroblast marker) (Sigma-Aldrich, catalog number: SCT059), dilution: 1:2,000

  6. Armenian hamster IgG-PE isotype control (BioLegend, catalog number: 400907), dilution: 1:200

  7. Rat IgG-Alexa Fluor 647 isotype control (BioLegend, catalog number: 400526), dilution: 1:200

  8. 4',6-diamidino-2-phenylindole (DAPI) 1 mg/mL (Life Technologies, catalog number: 62248), dilution: 1:500


Laboratory supplies

  1. 48-well clear TC-treated culture plate (Scientific Laboratory Supplies Ltd., catalog number: 3548)

  2. 96-well clear flat bottom TC-treated culture plate (Scientific Laboratory Supplies Ltd., catalog number: 3596)

  3. 24-well uncoated µ-plates (Ibidi, catalog number: 82421)

  4. Hydrophilic PTFE 12 mm cell culture inserts with 0.4 µm pores (Millipore, catalog number: PICM01250)

  5. Costar 5 mL stripette serological pipets (Scientific Laboratory Supplies Ltd., catalog number: 4487)

  6. Costar 10 mL stripette serological pipets (Scientific Laboratory Supplies Ltd., catalog number: 4488)

  7. Costar 25 mL stripette serological pipets (Scientific Laboratory Supplies Ltd., catalog number: 4489)

  8. Syringe 5 mL luer (VWR, catalog number: 613-2042)

  9. Swann-Morton surgical scalpels, No. 22 (Fisher Scientific Ltd., catalog number: 11758353)

  10. Sterile bijou 7 mL (Greiner Bio-One Ltd, catalog number: 189171)

  11. Weigh boats square 7 mL white (VWR, catalog number: 611-0093)

  12. Qualfast Zinc-plated steel washer M6 (Cromwell, catalog number: QFT6455541N)

  13. Petri dishes, 90 mm × 16 mm (Sarstedt, catalog number: 82.1472)

  14. 0.2 mL 8-strip non-flex PCR tubes (Starlab, catalog number: I1402-3700)

  15. Homogenisation tubes, screw cap micro tube 2 mL (Sarstedt, catalog number: 72.609)

  16. Homogenisation tube caps, standard screw cap (Starlab, catalog number: E1480-0100)

  17. Homogenisation beads, fastPrep-24TM lysing matrix D 1.4 mm ceramic spheres (MP Biomedicals, catalog number: 1169131050-CF)

  18. TapeStation loading tips (Agilent, catalog number: 5067-5153)

  19. TapeStation sample tube strips (Agilent, catalog number: 4014128)

  20. TapeStation sample tube caps (Agilent, catalog number: 4014125)

  21. MicroAmpTM fast optical 96-well reaction plate (ABI Applied Biosystems, catalog number: 4346907)

  22. MicroAmpTM optical adhesive film (ABI Applied Biosystems, catalog number: 4311971)

  23. Ice and ice box

  24. Laboratory tissue roll

Equipment

  1. Automated compresstome (Precisionary Instruments, catalog number: VF-510-0Z)

  2. Stainless steel blades for vibratome (Campden Instruments LTD, catalog number: 7550-1-SS)

  3. Leica SP8 inverted confocal microscope (Leica)

  4. SpectraMax® iD3 microplate reader (Molecular Devices, model: iD3, serial number: 37370-3811)

  5. FastPrep-24TM classic bead tissue homogeniser (MP Biomedicals, catalog number: 116004500)

  6. TapeStation 2200 (Agilent, catalog number: 18588)

  7. VeritiProTM thermal cycler (Applied Biosystems, catalog number: A48141)

  8. StepOnePlusTM real-time PCR system (Applied Biosystems, catalog number: 4376600)

  9. NanoDrop spectrophotometer (Thermo Scientific, catalog number: ND-1000)

  10. -20 °C freezer

  11. Incubator (humidified, 37 °C, 5% CO2) (Sanyo, catalog number: MCO-17A)

  12. Prism benchtop microfuge (Appleton Woods, catalog number: AA9760)

  13. Centrifuge vortex for PCR plate CVP-2 (Grant Bio, catalog number: CVP-2)

  14. IKA MS 3 basic shaker with MS 3.5 PCR plate attachment (IKA, catalog number: 0003617000)

  15. Curved dissecting forceps, 10 cm, Serr/C (World Precision Instruments Ltd, catalog number: 15915)

  16. Dissecting scissors, 10 cm, straight (World Precision Instruments Ltd, catalog number: 14393)

  17. Surgical scissors, 14 cm, straight (World Precision Instruments Ltd, catalog number: 14192)

  18. Spring scissors, 12 cm straight, 12 mm extra-fine blades (World Precision Instruments Ltd, catalog number: 14125)

  19. Metallic spatula (Fisher Scientific Ltd., catalog number: 11523482)

Software and datasets

  1. Leica Application Suite X software (Leica)

  2. Fiji (ImageJ, version 2.9)

  3. GraphPad Prism 10 (GraphPad)

  4. 2200 TapeStation System (Agilent, G2964AA)

  5. StepOne Software (Applied Biosystems)

Procedure

English
中文翻译

文章信息

版权信息

© 2024 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).

如何引用

Cheong, S. S., Luis, T. C., Hind, M. and Dean, C. H. (2024). A Novel Method for Floxed Gene Manipulation Using TAT-Cre Recombinase in Ex Vivo Precision-Cut Lung Slices (PCLS). Bio-protocol 14(8): e4980. DOI: 10.21769/BioProtoc.4980.

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分类

分子生物学 > DNA > 基因表达

细胞生物学 > 细胞工程 > 组织工程

细胞生物学 > 细胞成像 > 荧光

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