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The effects of CRISPR-Cas9 knockout of the TGF-β1 gene on antler cartilage cells in vitro.
Cellular & Molecular Biology Letters ( IF 8.3 ) Pub Date : 2019-06-22 , DOI: 10.1186/s11658-019-0171-z
Mingxiao Liu 1 , Xiangyu Han 1 , Hongyun Liu 1 , Danyang Chen 1 , Yue Li 1 , Wei Hu 1
Affiliation  

BACKGROUND Deer antler is the only mammalian organ that can be completely regenerated every year. Its periodic regeneration is regulated by multiple factors, including transforming growth factor β (TGF-β). This widely distributed multi-functional growth factor can control the proliferation and differentiation of many types of cell, and it may play a crucial regulatory role in antler regeneration. This study explored the role of TGF-β1 during the rapid growth of sika deer antler. METHODS Three CRISPR-Cas9 knockout vectors targeting the TGF-β1 gene of sika deer were constructed and packaged with a lentiviral system. The expression level of TGF-β1 protein in the knockout cell line was determined using western blot, the proliferation and migration of cartilage cells in vitro were respectively determined using EdU and the cell scratch test, and the expression levels of TGF-β pathway-related genes were determined using a PCR array. RESULTS Of the three gRNAs designed, pBOBI-gRNA2 had the best knockout effect. Knockout of TGF-β1 gene inhibits the proliferation of cartilage cells and enhances their migration in vitro. TGF-β signaling pathway-related genes undergo significant changes, so we speculate that when the TGF-β pathway is blocked, the BMP signaling pathway mediated by BMP4 may play a key role. CONCLUSIONS TGF-β1 is a newly identified regulatory factor of rapid growth in sika deer antler.

中文翻译:

CRISPR-Cas9敲除TGF-β1基因对体外鹿茸软骨细胞的影响。

背景技术鹿茸是唯一可以每年完全再生的哺乳动物器官。它的周期性再生受到多种因素的调节,包括转化生长因子β(TGF-β)。这种广泛分布的多功能生长因子可以控制多种细胞的增殖和分化,可能在鹿茸再生过程中发挥至关重要的调节作用。本研究探讨了TGF-β1在梅花鹿茸快速生长过程中的作用。方法构建三个靶向梅花鹿TGF-β1基因的CRISPR-Cas9敲除载体,并采用慢病毒系统进行包装。采用western blot检测敲除细胞系中TGF-β1蛋白的表达水平,采用EdU和细胞划痕试验分别检测软骨细胞体外增殖和迁移情况,并使用 PCR 阵列测定 TGF-β 通路相关基因的表达水平。结果在设计的三种gRNA中,pBOBI-gRNA2的敲除效果最好。敲除 TGF-β1 基因可抑制软骨细胞的增殖并增强其体外迁移。TGF-β信号通路相关基因发生显着变化,因此我们推测当TGF-β通路被阻断时,由BMP4介导的BMP信号通路可能起关键作用。结论 TGF-β1是新发现的梅花鹿茸快速生长调控因子。TGF-β信号通路相关基因发生显着变化,因此我们推测当TGF-β通路被阻断时,由BMP4介导的BMP信号通路可能起关键作用。结论 TGF-β1是新发现的梅花鹿茸快速生长调控因子。TGF-β信号通路相关基因发生显着变化,因此我们推测当TGF-β通路被阻断时,由BMP4介导的BMP信号通路可能起关键作用。结论 TGF-β1是新发现的梅花鹿茸快速生长调控因子。
更新日期:2019-11-01
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