Wilmot Jacob H, Warren Tracy L, Diniz Cassiano R A F, Carda Deger, Lafreniere Marrisa M, Nord Alex S, Wiltgen Brian J
Department of Psychology, University of California, Davis, Davis, CA, United States.
Center for Neuroscience, University of California, Davis, Davis, CA, United States.
Front Behav Neurosci. 2024 Dec 17;18:1500794. doi: 10.3389/fnbeh.2024.1500794. eCollection 2024.
Molecular and genetic techniques now allow selective tagging and manipulation of the population of neurons, often referred to as "engram cells," that were active during a specific experience. One common approach to labeling these cells is to use the transgenic mouse (TetTag). In addition to tagging cells active during learning, it is common to examine the reactivation of these cells using immediate early gene (IEG) expression as an index of neural activity. There are currently multiple TetTag lines available. The original line, cryopreserved at MMRRC, contains only the transgene, while Jackson Labs provides a version of the mouse that expresses both the and genes. In the current experiments, we examined IEG expression in these two mouse lines. Unexpectedly, we found that Jackson mice express increased levels of c-Fos in the hippocampus compared to wild type animals when examined with immunohistochemistry (IHC). The expression of other IEGs, such as Arc and Egr-1, was not elevated in these mice, suggesting that the overexpression of c-Fos is not the result of increased excitability or broad changes in gene expression. qPCR revealed that Jackson mice express mRNA corresponding to a c-Fos-Exon1-GFP fusion molecule, which may bind to C-Fos antibodies during IHC and inflate apparent c-Fos expression. Jackson mice did not differ from their wild-type counterparts in fear expression or memory, indicating no behavioral effect of the presence of a c-Fos-GFP fusion protein. These results identify a major limitation inherent in the use of Jackson mice.
分子和基因技术现在允许对在特定经历中活跃的神经元群体（通常称为“记忆印迹细胞”）进行选择性标记和操纵。标记这些细胞的一种常见方法是使用转基因小鼠（TetTag）。除了标记学习过程中活跃的细胞外，通常还使用即时早期基因（IEG）表达作为神经活动指标来检查这些细胞的重新激活情况。目前有多种TetTag品系可供使用。保存在MMRRC的原始品系仅包含转基因，而杰克逊实验室提供了一种同时表达 和 基因的小鼠版本。在当前实验中，我们检查了这两种小鼠品系中的IEG表达。出乎意料的是，我们发现，当用免疫组织化学（IHC）检测时，与野生型动物相比，杰克逊 小鼠海马体中c-Fos的表达水平升高。在这些小鼠中，其他IEG（如Arc和Egr-1）的表达并未升高，这表明c-Fos的过表达不是兴奋性增加或基因表达广泛变化的结果。定量聚合酶链反应（qPCR）显示，杰克逊 小鼠表达与c-Fos-Exon1-GFP融合分子相对应的信使核糖核酸（mRNA），该分子在免疫组织化学过程中可能与C-Fos抗体结合，从而夸大了明显的c-Fos表达。杰克逊 小鼠与野生型小鼠在恐惧表达或记忆方面没有差异，这表明c-Fos-GFP融合蛋白的存在没有行为学影响。这些结果确定了使用杰克逊 小鼠存在的一个主要局限性。
