Gene Expression profiling of transgenic mice expressing the genetically encoded calcium indicator TN-XXL in muscle and brain tissues

Engineering of genetically encoded calcium indicators predominantly focused on optimizing fluorescence changes, but effects of indicator expression on host organisms have largely not been addressed. Here, we report biocompatibility and wide-spread functional expression of the genetically encoded calcium indicator TN-XXL in a transgenic mouse model. To validate the model and to characterize potential effects of indicator expression we assessed both indicator function and a variety of host parameters such as anatomy, physiology, behavior and gene expression profiles in these mice. We also demonstrate the usefulness of primary cell types and organ explants prepared from these mice for imaging applications. While we do find mild signatures of indicator expression that may guide further indicator development the "green" indicator mice generated provide a well characterized resource of primary cells and tissues for in vitro and in vivo calcium imaging applications. Using expression profiling we could detect a number of transcripts regulated predominantly in heart and skeletal muscle of TN-XXL transgenic mice but only very few showed a level of regulation of a factor of two or higher and thus the majority of these changes were considered physiologically irrelevant. Remarkably this regulation occurred in skeletal and cardiac muscle, but not in brain. This suggests that skeletal muscle TnC as calcium sensing moiety within the indicator may be bio-orthogonal in brain but may lose some of this advantage in muscle tissue where it is derived from. Factorial design comparing transgenic mice with wild type littermates in three different tissues (hippocampus, skeletal and heart muscle)绝大多数基因编码钙指示剂（genetically encoded calcium indicators）的工程改造均以优化荧光变化为核心目标，但此类指示剂的表达对宿主生物体的影响，在很大程度上未得到充分研究。本研究报道了基因编码钙指示剂TN-XXL在转基因小鼠模型中的生物相容性与广泛功能性表达。为验证该模型并表征指示剂表达的潜在影响，我们对该类小鼠的指示剂功能及多项宿主参数进行了评估，涵盖解剖学特征、生理学指标、行为表现与基因表达谱等维度。此外，我们还证实了从该类小鼠中制备的原代细胞与器官外植体在成像应用中的实用价值。尽管我们观测到了可指导后续指示剂开发的轻度表达特征，但本研究构建的‘绿色’指示剂转基因小鼠，可为体外（in vitro）与体内（in vivo）钙成像应用提供一套经过充分表征的原代细胞与组织资源。通过表达谱分析，我们在TN-XXL转基因小鼠的心肌与骨骼肌中检测到了一批受调控的转录本，但其中仅极少数的调控倍数达到2倍及以上，因此绝大多数此类变化被认为无生理学意义。值得注意的是，此类调控仅发生在骨骼肌与心肌中，脑组织中并未出现此类变化。这表明，作为指示剂内钙感应结构域的骨骼肌肌钙蛋白C（TnC），在脑组织中或许具备生物正交性，但在其来源的肌肉组织中则可能丧失部分此类优势。本研究采用析因设计，针对三种不同组织（海马体（hippocampus）、骨骼肌与心肌）中的转基因小鼠与其野生型同窝仔鼠进行了对比分析。