National Heart and Lung Institute (NHLI), Imperial College London, London SW7 2AZ, UK.
Centre for Inflammatory Diseases, Department of Immunology and Inflammation, Imperial College London, London W12 0NN, UK.
Dis Model Mech. 2023 Nov 1;16(11). doi: 10.1242/dmm.050267. Epub 2023 Nov 3.
Precision-cut lung slices (PCLS) are used for a variety of applications. However, methods to manipulate genes in PCLS are currently limited. We developed a new method, TAT-Cre recombinase-mediated floxed allele modification in tissue slices (TReATS), to induce highly effective and temporally controlled gene deletion or activation in ex vivo PCLS. Treatment of PCLS from Rosa26-flox-stop-flox-EYFP mice with cell-permeant TAT-Cre recombinase induced ubiquitous EYFP protein expression, indicating successful Cre-mediated excision of the upstream loxP-flanked stop sequence. Quantitative real-time PCR confirmed induction of EYFP. We successfully replicated the TReATS method in PCLS from Vangl2flox/flox mice, leading to the deletion of loxP-flanked exon 4 of the Vangl2 gene. Cre-treated Vangl2flox/flox PCLS exhibited cytoskeletal abnormalities, a known phenotype caused by VANGL2 dysfunction. We report a new method that bypasses conventional Cre-Lox breeding, allowing rapid and highly effective gene manipulation in ex vivo tissue models.
精准切割肺切片（PCLS）被广泛应用于多种领域。然而，目前用于 PCLS 基因操作的方法还很有限。我们开发了一种新的方法，TAT-Cre 重组酶介导的组织切片中 floxed 等位基因修饰（TReATS），用于在离体 PCLS 中诱导高效且具有时间控制的基因缺失或激活。用细胞通透性 TAT-Cre 重组酶处理 Rosa26-flox-stop-flox-EYFP 小鼠的 PCLS，诱导广泛表达 EYFP 蛋白，表明 Cre 介导的上游 loxP 侧翼的终止序列的有效切除。实时定量 PCR 证实了 EYFP 的诱导。我们成功地在 Vangl2flox/flox 小鼠的 PCLS 中复制了 TReATS 方法，导致 Vangl2 基因loxP 侧翼的外显子 4 缺失。用 Cre 处理的 Vangl2flox/flox PCLS 表现出细胞骨架异常，这是 VANGL2 功能障碍的已知表型。我们报告了一种新的方法，绕过了传统的 Cre-Lox 繁殖，允许在离体组织模型中快速且高效地进行基因操作。
