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ScRNAseq analysis of lung with eliminating p16h cells [scRNA-seq Lung_DTR]

ABSTRACT: We employed the Chromium GEM-X single-cell RNA sequencing platform by 10x Genomics to investigate how the removal of p16-high-expressing cells affects the lung single-cell transcriptome of 22-month-old male mice. In this experiment, we utilized p16-CreERT2/R26-LSL-DTR mice, administering tamoxifen and diphtheria toxin at 21 months of age. Lung tissues were harvested two weeks later for analysis. Our objective was to examine how the elimination of p16-high cells influences intercellular interactions among diverse cell types in aged lungs.

ORGANISM(S): Mus musculus

PROVIDER: GSE300530 | GEO | 2025/07/02

REPOSITORIES: GEO

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Project description:Injury of the lung epithelium is one of the initial events driving acute lung diseases such as acute lung injury and acute respiratory distress syndrome. Repeated epithelial injury followed by aberrant repair leads to fibroblast activation and extracellular matrix deposition and is therefore considered as the major cause of chronic pulmonary diseases, such as idiopathic pulmonary fibrosis. In vitro models as well as in vivo animal models are necessary to investigate early disease mechanisms and altered signalling pathways of epithelial cells. One way to target specific cells is the diphtheria toxin receptor/diphtheria toxin (DTR/DT) system. The human DTR (hDTR) is sensitive to DT, thereby inducing cell death by blocking protein synthesis, while the murine DTR is at least 10^5 times more resistant to DT. Consequently, expression of the hDTR and application of DT lead to targeted cell depletion. Therefore, a novel and flexible DTR/DT model of acute epithelial lung injury was established and described recently, which is driven by adeno-associated virus (AAV) variant 6.2 mediated hDTR expression. The AAV6.2 vector transduces specifically into bronchial epithelial and alveolar epithelial type II cells leading to their depletion after DT administration. In this study we analysed the bulk lung proteome of the recently established AAV-DTR/DT mouse model of acute epithelial lung injury, providing a detailed insight into proteomic changes upon injury. Frozen pulverized lung tissue samples (~10 mg per sample) from AAV-stuffer control (1 E11 viral genome, n=7) and AAV-hDTR (0.3 E11 viral genome, n=7) treated mice 24 h after intratracheal application of 100 ng DT were analysed in a TMTpro-based workflow. TMTpro-labelled peptides were fractionated into 24 fractions using off-line high pH reversed phase chromatography. Fractions were analysed on an Orbitrap Eclipse Tribrid mass spectrometer in combination with the FAIMS Pro Interface (Thermo Scientific) using a SPS-MS3 based method including real-time search.

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ScRNAseq analysis of lung with eliminating p16h cells [scRNA-seq Lung_DTR]

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