Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors.
Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors.
复制标题
使用腺相关病毒载体产生产光体细胞转基因小鼠。
DOI：
10.1038/s41598-020-59075-3
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发表时间：
2020
影响因子：
4.6
通讯作者：
Karda R
中科院分区：
文献类型：
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作者：
Karda R
我们以前设计了一个慢病毒载体库，以产生体细胞转基因啮齿动物，在有意识的，自由移动的啮齿动物中，使用全身生物发光成像来监测患病器官中的信号通路。我们现在已经将这项技术扩展到腺相关病毒载体。我们首先通过评估新生儿静脉内递送AAV 8后的GFP表达来探索生物分布。我们观察到广泛的基因表达，中枢和外周神经系统，肝脏，肾脏和骨骼肌。接下来，我们选择了组成型SFFV启动子和NFκB结合序列用于生物发光和生物传感器评估。静脉内注射含有萤火虫荧光素酶和eGFP的AAV 8在任一元件的转录控制下导致强烈和持久的广泛荧光素酶表达。单剂量LPS诱导AAV 8-NFκB小鼠荧光素酶表达增加10倍，免疫组织化学显示星形胶质细胞和神经元形态的细胞中有GFP表达。重要的是，全身生物发光持续长达240天。我们通过使用NFκB反应元件在AAV系统中验证了一种新的生物传感器技术，并揭示了其在LPS诱导的炎症模型中以非侵入性方式监测信号传导途径的潜力。该技术补充了现有的生殖系转基因模型，并可能适用于其他啮齿动物疾病模型。
We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.