Transcriptome analysis of tendon in Mkx conditional knock out mice. Transcriptome analysis of tendon in Mkx conditional knock out mice

Tendons are crucial connective tissues that link muscle to bone. We have demonstrated that the transcription factor Mkx plays a pivotal role in tendon development and homeostasis by regulating collagen fibril organization through tendon cell fate determination from progenitor cells and promoting the expression of type I collagen and proteoglycans. However, the precise role of Mkx in tendon homeostasis after birth is not well understood. Therefore, we generated conditional Mkx knockout mice by crossing Mkx-CreER mice with Mkx floxed mice. Following tamoxifen administration, we performed transcriptome analysis of tendons from control mice (Mkx-CreER/+ and Mkx flox/+) and Mkx conditional knockout mice (Mkx-CreER/flox) Overall design: Mkx-CreER/+, Mkx-flox, and Mkx-CreER/flox mice were administered tamoxifen at 6 weeks of age. After 4 weeks, the Achilles tendon was harvested, and total RNA was purified using the Direct-zol RNA MicroPrep Kit (Zymo Research). RNA-seq libraries were prepared using the GenNext RamDA-seq Single Cell Kit RMD-101 (TOYOBO), and the libraries were sequenced on a NovaSeq 6000 (Illumina).

肌腱是连接肌肉与骨骼的关键结缔组织。本研究证实，转录因子（transcription factor）Mkx可通过调控胶原纤维（collagen fibril）排布——具体包括从祖细胞（progenitor cells）中定向决定腱细胞命运，并促进I型胶原（type I collagen）与蛋白聚糖（proteoglycans）的表达——在肌腱发育与稳态维持中发挥关键作用。然而，出生后Mkx在肌腱稳态维持中的精确功能尚未被充分阐明。为此，我们通过将Mkx-CreER小鼠与Mkx floxed小鼠杂交，构建了条件性Mkx敲除小鼠模型。在给予他莫昔芬（tamoxifen）处理后，我们分别对对照组小鼠（基因型为Mkx-CreER/+与Mkx flox/+）及Mkx条件性敲除小鼠（基因型为Mkx-CreER/flox）的肌腱样本开展转录组（transcriptome）分析。实验整体设计：于6周龄时对Mkx-CreER/+、Mkx-flox及Mkx-CreER/flox小鼠注射他莫昔芬；4周后采集其跟腱（Achilles tendon）组织，使用Direct-zol RNA MicroPrep试剂盒（Zymo Research公司）提取总RNA（total RNA）；采用GenNext RamDA-seq单细胞试剂盒RMD-101（TOYOBO公司）构建RNA测序文库，并依托NovaSeq 6000测序平台（Illumina公司）完成测序。