Generation and Characterization of a Cell Type-Specific, Inducible Cre-Driver Line to Study Olfactory Processing.
Generation and Characterization of a Cell Type-Specific, Inducible Cre-Driver Line to Study Olfactory Processing.
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DOI：
10.1523/jneurosci.3076-20.2021
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发表时间：
2021-07-28
期刊：
影响因子：
--
通讯作者：
Fukunaga I
中科院分区：
文献类型：
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作者：
Koldaeva A;Zhang C;Huang YP;Reinert JK;Mizuno S;Sugiyama F;Takahashi S;Soliman T;Matsunami H;Fukunaga I
在大脑的感觉系统中，存在从刺激中提取不同特征以产生各种行为库的机制。这些通常对应于感觉处理的不同阶段的不同细胞类型。在哺乳动物的嗅觉系统中，复杂的信息处理始于嗅球，其输出由僧帽细胞（MC）和簇状细胞（TC）传达。尽管它们之间存在许多差异，尽管它们在信息层次结构中占据关键地位，但区分它们的Cre-driver线还不存在。在这里，我们试图鉴定在小鼠的MC和TC之间差异表达的基因，最终目标是从使用大型且公开可用的单细胞RNA-seq数据集的转录组分析开始，生成细胞类型特异性的Cre-driver系。许多基因的差异表达，但只有少数表现出一致的表达在MC和所需的特异性。在使用ISH进一步验证这些推定的标记之后，两个基因（即，Pkib和Lbdh 2）仍然是有希望的候选人。使用CRISPR/Cas9介导的基因编辑，我们生成了Cre驱动系，并分析了产生的重组模式。这表明我们的新诱导型Cre-driver系Lbhd 2-CreERT 2可用于以他莫昔芬剂量依赖性方式遗传标记雄性和雌性小鼠中的MC，如通过索马体位置、投射模式和体内感觉诱发反应所评估的那样。因此，这是一个很有前途的工具，用于研究细胞类型对嗅觉处理的特定贡献，并展示了公开获取的数据在加速科学发展方面的力量。在大脑中，不同的细胞类型发挥着独特的作用。因此，重要的是要有专门研究独特细胞类型的工具。对于哺乳动物的嗅觉，信息首先由嗅球的回路处理，其中两种类型的细胞，僧帽细胞和簇状细胞，输出不同的信息。我们产生了一个转基因小鼠系，使二尖瓣细胞被特异性标记或操纵。这是通过使用大型公共基因表达数据集寻找对二尖瓣细胞特异性的基因，并使用基因编辑技术CRISPR/Cas9创建转基因小鼠来实现的。这将使科学家能够更好地研究嗅觉背后的并行信息处理。
In sensory systems of the brain, mechanisms exist to extract distinct features from stimuli to generate a variety of behavioral repertoires. These often correspond to different cell types at various stages in sensory processing. In the mammalian olfactory system, complex information processing starts in the olfactory bulb, whose output is conveyed by mitral cells (MCs) and tufted cells (TCs). Despite many differences between them, and despite the crucial position they occupy in the information hierarchy, Cre-driver lines that distinguish them do not yet exist. Here, we sought to identify genes that are differentially expressed between MCs and TCs of the mouse, with an ultimate goal to generate a cell type-specific Cre-driver line, starting from a transcriptome analysis using a large and publicly available single-cell RNA-seq dataset. Many genes were differentially expressed, but only a few showed consistent expressions in MCs and at the specificity required. After further validating these putative markers using ISH, two genes (i.e., Pkib and Lbdh2) remained as promising candidates. Using CRISPR/Cas9-mediated gene editing, we generated Cre-driver lines and analyzed the resulting recombination patterns. This indicated that our new inducible Cre-driver line, Lbhd2-CreERT2, can be used to genetically label MCs in a tamoxifen dose-dependent manner, both in male and female mice, as assessed by soma locations, projection patterns, and sensory-evoked responses in vivo. Hence, this is a promising tool for investigating cell type-specific contributions to olfactory processing and demonstrates the power of publicly accessible data in accelerating science. SIGNIFICANCE STATEMENT In the brain, distinct cell types play unique roles. It is therefore important to have tools for studying unique cell types specifically. For the sense of smell in mammals, information is processed first by circuits of the olfactory bulb, where two types of cells, mitral cells and tufted cells, output different information. We generated a transgenic mouse line that enables mitral cells to be specifically labeled or manipulated. This was achieved by looking for genes that are specific to mitral cells using a large and public gene expression dataset, and creating a transgenic mouse using the gene editing technique, CRISPR/Cas9. This will allow scientists to better investigate parallel information processing underlying the sense of smell.
影响因子：
3
作者：
Hovis, Kenneth R.;Padmanabhan, Krishnan;Urban, Nathaniel N.
通讯作者：
Urban, Nathaniel N.