PDF(Pubmed)
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.
摘要：
精确切割肺切片（PCLS），离体3D肺组织模型，已广泛用于肺部研究中的各种应用。PCLS充当体外和体内模型之间的极好中介,因为它们将所有驻留细胞类型保留在其天然生态位内,同时保留细胞外基质环境。该方案描述了TReATS（组织切片中TAT-Cre重组酶介导的Floxed等位基因修饰）方法，该方法能够在源自成年Floxed动物的PCLS中进行快速有效的基因修饰。这里,我们提出了TReATS方法的详细协议，由两个简单步骤组成:PCLS生成和在TAT-Cre重组酶溶液中孵育。基因修饰的后续验证涉及PCLS的活染色和成像，实时定量PCR,和细胞活力评估。这个为期四天的协议消除了复杂的Cre繁殖的需要，规避与基因突变相关的过早致死问题，并大大减少了动物的使用。TReATS方法为复杂的离体组织模型中的基因修饰提供了简单且可重复的解决方案，加快基因功能的研究，疾病机制,以及药物靶点的发现。关键特征•在四天内在复杂的基于组织的模型中实现永久性离体基因修饰。•可应用于诱导基因缺失或激活的高度适应性基因修饰方法。•允许在受控离体设置中进行简单的Cre剂量测试,其优点是使用从与真实对照相同的动物产生的PCLS。•通过优化，该方法可应用于其他器官的精确切割组织切片。
