导管内可视化通过NF-κB记者的转基因小鼠模型的乳腺炎：信号注射LPS
Summary
这里描述的是一种技术，其中脂多糖注入泌乳小鼠乳腺通过乳头模拟乳腺炎，通常由细菌感染引起的一个条件。脂多糖（LPS）的结果中增加核因子kappa B（NF-κB）信号，通过NF-κB荧光素酶报告基因小鼠的生物发光成像的可视化。
Abstract
疾病进展和相关的机制，严格学习阶段，最终的临床前模型测试干预是必要的，人类疾病的动物模型。在这些方法中，我们描述了一种技术，其中脂多糖（LPS）被注入到通过乳头，有效建模乳腺炎，或炎症，腺体泌乳小鼠乳腺。增加核因子kappa B（NF-κB）信号，这个模拟感染的结果，显示通过生物 发光成像的NF-κB荧光素酶报告基因小鼠1。
我们的最终目标是研究开发这些方法有关的炎症，乳腺炎在哺乳期腺，通常包括发红，肿胀，免疫细胞浸润2,3。因此，我们敏锐地意识到，切口或任何类型的受伤的皮肤，乳头，或腺体介绍LPS可以在我们的方法中不能使用，因为该方法可能会混淆的读出的炎症。我们也想要一个直接的方法，并不需要特制的手绘移液器或利用显微操作器来保存这些专门的工具。因此，我们确定了一个完整的乳头进入乳腺导管，使用市售的胰岛素注射器和注射剂。此方法是成功的，并允许我们研究与LPS注射相关的炎症，由注射的过程中没有任何额外的效果重叠。此外，该方法还利用NF-κB的荧光素酶报告基因的转基因小鼠和生物发光成像技术的视觉和定量显示增加的NF-κB信号在LPS注射压盖4。
谁愿意在哺乳期乳腺疾病模型，因为归根结底，这项技术的许多学科的研究人员感兴趣的是这些方法这里描述的可用于注射的物质的数量，并不限定于仅LPS。
Protocol
所有的动物实验，范德比尔特大学实验动物护理和使用委员会的批准。 1。准备转基因小鼠 NGL（N F-κB-G FP-L uciferase）记者转基因（前面描述1）阳性的雌性小鼠，用于此过程。当女性的年龄至少6周，交配与FVB野生型男性。后宫养殖可以使用（在一个笼子里，男1例，女4例）。 （请注意：FVB应变小鼠用于目前的研究中，也可以使用较深的菌株如C57BL6?…
Discussion
在这里，我们利用转基因NF-κB记者的鼠标和优化注射技术模型乳腺炎，乳房组织的炎症最常见的原因是细菌感染。在此过程中最关键的一步是导管内注射本身。
在实践中，这通常也是显而易见注射导管内是否已经成功。注射剂（LPS或PBS）应该感到流体，应该有很少或几乎没有阻力的注射器被清空。最常见的陷阱是导致阻塞通道进入腺体，以乳头的损伤。这可以是可视化的寻?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院资助CA113734到F Yull和退伍军人事务部（TS布莱克韦尔）获得。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Lipopolysaccharide from Escherichia coli (LPS) |
Sigma | L 2880 | serotype 055:B5 |
| PBS | Mediatech, Inc. | 46-013-CM | |
| 31 Gauge insulin syringe | Becton Dickinson | 328438 | Short needle |
| Forceps | World Precision Instruments | 15908 | |
| Dissecting scope | Leica | M3Z | |
| Terrell Isoflurane, USP | MINRAD INC. for Rx Elite | NDC 66794-011-25 | |
| IVIS 200 imaging system | Caliper (formerly Xenogen) | N/A | |
| Syringe for luciferin injection | Becton Dickinson | 309597 | 1cc; 26G5/8 |
| D-Luciferin Firefly, sodium salt monohydrate (Luciferin substrate) |
Biosynth Chemistry and Biology | L-8240 | Dilute to 10 mg/ml |
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Tags
Intraductal InjectionLPSMouse ModelMastitisNF-κB ReporterTransgenic AnimalDisease ProgressionPre-clinical ModelsLipopolysaccharideLactating Mouse Mammary GlandSimulated InfectionInflammationNuclear Factor Kappa BBioluminescent ImagingLuciferase Reporter MouseRednessSwellingImmune Cell InfiltrationStraight-forward MethodInsulin SyringeMammary Duct
Citer Cet Article
Barham, W., Sherrill, T., Connelly, L., Blackwell, T. S., Yull, F. E. Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic. J. Vis. Exp. (67), e4030, doi:10.3791/4030 (2012).