Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, 35131 Padua, Italy.
Neuroscience Institute, Italian National Research Council (CNR), Via U. Bassi 58/B, 35131 Padua, Italy.
Function (Oxf). 2021 Feb 25;2(3):zqab012. doi: 10.1093/function/zqab012. eCollection 2021.
Mitochondria play a key role in cellular calcium (Ca) homeostasis. Dysfunction in the organelle Ca handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca indicators (GECIs) have been developed to study Ca dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a -STOP- sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca imaging experiments performed and in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca concentration ([Ca]).
线粒体在细胞钙离子（Ca）稳态中发挥着关键作用。细胞器 Ca 处理功能障碍似乎与多种病理状况有关，从神经退行性疾病、心力衰竭到恶性转化。在过去的几年中，已经开发出几种基于靶向绿色荧光蛋白（GFP）的基因编码 Ca 指示剂（GECI），用于研究活细胞线粒体内部的 Ca 动力学。令人惊讶的是，虽然有许多表达不同类型胞质 GECI 的转基因小鼠，但表达定位于线粒体的 GECI 的例子很少，而且没有一个具有足够的空间分辨率。在这里，我们报告了一种转基因小鼠系（以下称为 mt-Cam）的产生和特征，用于控制表达一种靶向线粒体、基于Förster 共振能量转移（FRET）的 Cameleon，即 4mtD3cpv。为了实现这一目标，我们通过插入优化的 4mtD3cpv 序列（前面带有 -STOP- 序列）来设计小鼠基因组位点。通过 Cre 重组酶介导的切除，可以以单交叉的方式轻易地以组织特异性方式表达该探针。在普遍表达 Cre 的情况下，Cameleon 特异性定位于分析的所有器官和组织的细胞线粒体基质中，从胚胎到老年动物。在脑切片中进行的 Ca 成像实验证实了探针在分离细胞和活体组织中的功能。这种新的转基因小鼠系允许在不同组织中进行线粒体 Ca 动力学研究，而无需进行侵入性干预（如病毒感染或电穿孔），这可能允许简单地根据线粒体 Ca 浓度 ([Ca]) 对荧光信号进行校准。
