Molecular Nociception Group, Wolfson Institute for Biomedical Research, University College London, UK.
Mol Pain. 2011 Dec 21;7:100. doi: 10.1186/1744-8069-7-100.
Tissue-specific gene deletion has proved informative in the analysis of pain pathways. Advillin has been shown to be a pan-neuronal marker of spinal and cranial sensory ganglia. We generated BAC transgenic mice using the Advillin promoter to drive a tamoxifen-inducible CreERT2 recombinase construct in order to be able to delete genes in adult animals. We used a floxed stop ROSA26LacZ reporter mouse to examine functional Cre expression, and analysed the behaviour of mice expressing Cre recombinase.
We used recombineering to introduce a CreERT2 cassette in place of exon 2 of the Advillin gene into a BAC clone (RPCI23-424F19) containing the 5' region of the Advillin gene. Transgenic mice were generated using pronuclear injection. The resulting AvCreERT2 transgenic mice showed a highly specific expression pattern of Cre activity after tamoxifen induction. Recombinase activity was confined to sensory neurons and no expression was found in other organs. Less than 1% of neurons showed Cre expression in the absence of tamoxifen treatment. Five-day intraperitoneal treatment with tamoxifen (2 mg per day) induced Cre recombination events in ≈90% of neurons in dorsal root and cranial ganglia. Cell counts of dorsal root ganglia (DRG) from transgenic animals with or without tamoxifen treatment showed no neuronal cell loss. Sensory neurons in culture showed ≈70% induction after 3 days treatment with tamoxifen. Behavioural tests showed no differences between wildtype, AvCreERT2 and tamoxifen-treated animals in terms of motor function, responses to light touch and noxious pressure, thermal thresholds as well as responses to inflammatory agents.
Our results suggest that the inducible pan-DRG AvCreERT2 deleter mouse strain is a useful tool for studying the role of individual genes in adult sensory neuron function. The pain phenotype of the Cre-induced animal is normal; therefore any alterations in pain processing can be unambiguously attributed to loss of the targeted gene.
组织特异性基因缺失已被证明在疼痛通路分析中具有重要意义。Advillin 已被证明是脊髓和颅感觉神经节的全神经元标志物。我们使用 Advillin 启动子生成 BAC 转基因小鼠，以驱动可诱导的 tamoxifen 的 CreERT2 重组酶构建体，从而能够在成年动物中删除基因。我们使用 floxed 停止 ROSA26LacZ 报告小鼠来检查功能性 Cre 表达，并分析表达 Cre 重组酶的小鼠的行为。
我们使用重组酶引入 CreERT2 盒替代 Advillin 基因的外显子 2，该基因位于 Advillin 基因的 5'区域的 BAC 克隆（RPCI23-424F19）中。通过核注射生成转基因小鼠。在用 tamoxifen 诱导后，所得的 AvCreERT2 转基因小鼠显示出 Cre 活性的高度特异性表达模式。重组酶活性仅限于感觉神经元，在其他器官中没有表达。在没有 tamoxifen 处理的情况下，不到 1%的神经元显示 Cre 表达。用 2mg/天的 tamoxifen 进行 5 天的腹腔内处理，诱导背根和颅神经节中约 90%的神经元发生 Cre 重组事件。有或没有 tamoxifen 处理的转基因动物的背根神经节 (DRG) 的细胞计数显示神经元没有丢失。用 tamoxifen 处理 3 天后，培养的感觉神经元的诱导率约为 70%。行为测试显示，野生型、AvCreERT2 和用 tamoxifen 处理的动物在运动功能、对轻触和有害压力的反应、热阈值以及对炎症剂的反应方面没有差异。
我们的结果表明，可诱导的全 DRG AvCreERT2 缺失小鼠品系是研究单个基因在成年感觉神经元功能中的作用的有用工具。Cre 诱导动物的疼痛表型正常；因此，任何疼痛处理的改变都可以明确归因于靶向基因的缺失。
