Aging and Metabolism Research Program, Oklahoma Medical Research Foundation; Genes & Human Disease Research Program, Oklahoma Medical Research Foundation; Oklahoma City Veterans Affairs Medical Center.
Aging and Metabolism Research Program, Oklahoma Medical Research Foundation; Oklahoma City Veterans Affairs Medical Center.
J Vis Exp. 2023 Feb 24(192). doi: 10.3791/64765.
Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.
评估细胞类型特异性的表观基因组和转录组变化是理解卵巢衰老的关键。为此，优化了翻译核糖体亲和纯化（TRAP）方法和特定细胞类型标记核（INTACT）方法，以便随后使用新型转基因 NuTRAP 小鼠模型对卵巢细胞特异性转录组和表观基因组进行配对研究。NuTRAP 等位基因的表达受 floxed STOP 盒的控制，并且可以使用启动子特异性 Cre 线靶向特定的卵巢细胞类型。由于最近的研究表明卵巢基质细胞在驱动早衰表型中起作用，因此使用 Cyp17a1-Cre 驱动子将 NuTRAP 表达系统靶向到基质细胞。NuTRAP 构建的诱导特异性地针对卵巢基质成纤维细胞，并且可以从单个卵巢中获得足够用于测序研究的 DNA 和 RNA。这里提出的 NuTRAP 模型和方法可用于研究任何具有可用 Cre 线的卵巢细胞类型。
