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一个Cre-依赖的记者鼠标用于蛋白激酶A活动动态的定量实时成像

Elizabeth I Tilden^1,2, Aditi Maduskar¹, Anna Oldenborg¹

¹Department of Neuroscience, Washington University in St. Louis, St. Louis, MO, United States.

bioRxiv : the preprint server for biology

|November 14, 2023

概括

我们开发了FL-AK记者小鼠,用于实时成像蛋白激酶A (PKA) 活动. 这种强大的工具克服了以前方法的局限性,使各种细胞类型的PKA动态研究能够精确.

科学领域:

分子生物学分子生物学
细胞信号传输细胞信号传输
神经科学是一个神经科学.

背景情况:

细胞内信号传递动力学对细胞功能至关重要,蛋白激酶A (PKA) 在各种过程中发挥关键作用,如新陈代谢,大脑活动,发育和癌症.
以前用于量化PKA活性的方法,如FLIM-AKAR报告员,依赖于侵入性传递技术 (病毒感染,电穿孔),导致变量表达和针对特定细胞类型的限制.
需要一种更强大,更一致,更灵活的方法来在体内可视化PKA活动动态.

研究的目的:

开发一个新的记者鼠标线,FL-AK,用于FLIM-AKAR记者的Cre-dependent表达.
建立一种可靠和一致的方法,用于在体内对PKA活性进行定量成像.
为了使在不同细胞类型和生理上下文中研究PKA信号动态.

主要方法:

通过将FLIM-AKAR集成到ROSA26位点以Cre-依赖的方式生成FL-AK记者鼠标系.
在激活Gαs和Gαq结合受体时,在脑切片中验证FLIM-AKAR表达和PKA活性报告.
在体内评估PKA酸化动态响应神经调节器受体激活.

主要成果:

随着时间的推移,FL-AK鼠标系列提供了强大而一致的FLIM-AKAR表达.
在脑切片中,FL-AK成功地报告了对Gαs和Gαq结合受体激活的反应中增加的PKA活性.
在体内研究表明,FL-AK能够在神经调节器受体激活后跟踪PKA酸化变化.

结论:

FL-AK 记者鼠标提供了一种定量,强大和灵活的工具,用于研究PKA信号动态.
这种小鼠线克服了以前的输送方法的局限性,允许精确地研究PKA在各种细胞类型中的活性.
FL-AK促进了PKA活动的实时监测,促进了我们对其在生理和病理过程中的作用的理解.

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