Protocol for auxin-inducible depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells.
Protocol for auxin-inducible depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells.
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DOI：
10.1016/j.xpro.2023.102644
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发表时间：
2023-12-15
期刊：
影响因子：
--
通讯作者：
Makeyev, Eugene, V
中科院分区：
文献类型：
--
作者：
Kainov, Yaroslav;Zhuravskaya, Anna;Makeyev, Eugene, V
诱导降解感兴趣的蛋白质为功能研究提供了一种强有力的方法。在这里，我们提出了一种在小鼠胚胎干细胞（ESCs）中严格控制rna结合蛋白PTBP1耗竭的方案。我们描述了建立表达多西环素诱导的生长素受体蛋白OsTIR1的ESC系和使用CRISPR-Cas9和同源定向修复试剂标记内源性pptp1等位基因的步骤。然后，我们详细介绍了通过免疫印迹法测定诱导pptbp1敲低效率的程序。该方案适用于其他蛋白质靶点。有关该协议的使用和执行的完整细节，请参阅Iannone等人。用生长素诱导的降解序列标记感兴趣基因的两个等位基因的方案重组克隆基因分型和测定标记蛋白降解的建议出版者注：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指导方针。诱导降解感兴趣的蛋白质为功能研究提供了一种强有力的方法。在这里，我们提出了一种在小鼠胚胎干细胞（ESCs）中严格控制rna结合蛋白PTBP1耗竭的方案。我们描述了建立表达多西环素诱导的生长素受体蛋白OsTIR1的ESC系和使用CRISPR-Cas9和同源定向修复试剂标记内源性pptp1等位基因的步骤。然后，我们详细介绍了通过免疫印迹法测定诱导pptbp1敲低效率的程序。该方案适用于其他蛋白质靶点。
Inducible degradation of proteins of interest provides a powerful approach for functional studies. Here, we present a protocol for tightly controlled depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells (ESCs). We describe steps for establishing an ESC line expressing doxycycline-inducible auxin receptor protein OsTIR1 and tagging endogenous Ptbp1 alleles using CRISPR-Cas9 and homology-directed repair reagents. We then detail procedures for assaying the efficiency of inducible PTBP1 knockdown by immunoblotting. This protocol is adaptable for other protein targets. For complete details on the use and execution of this protocol, please refer to Iannone et al. Efficient knock-in of a Dox-inducible OsTIR1 gene into mouse embryonic stem cell genome Protocol for tagging both alleles of a gene of interest with auxin-inducible degron sequences Recommendations for genotyping recombinant clones and assaying tagged protein degradation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Inducible degradation of proteins of interest provides a powerful approach for functional studies. Here, we present a protocol for tightly controlled depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells (ESCs). We describe steps for establishing an ESC line expressing doxycycline-inducible auxin receptor protein OsTIR1 and tagging endogenous Ptbp1 alleles using CRISPR-Cas9 and homology-directed repair reagents. We then detail procedures for assaying the efficiency of inducible PTBP1 knockdown by immunoblotting. This protocol is adaptable for other protein targets.
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影响因子：
16.6
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作者：
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通讯作者：
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影响因子：
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