IL-22表达淋巴细胞使用记者小鼠的可视化
Summary
We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.
Abstract
报告小鼠已被广泛用于观察目标基因的表达的定位。该协议的重点是建立一个新的转基因记者小鼠模型的战略。我们选择了可视化白介素（IL）22的基因表达，因为这种细胞因子具有在肠道内，在那里它有助于修理由炎症损伤组织的重要活动。记者系统提供优于识别产品在体内的其它方法相当大的优势。在IL-22的情况下，其他的研究已经首次分离自组织的细胞，然后再刺激在体外细胞中。 IL-22，这通常是分泌的，使用一种药物被困在细胞内，并用于细胞内染色来可视化它。本方法识别能够产生的IL-22的细胞，但并不确定它们是否在体内做。记者设计包括以这样的洼插入基因为荧光蛋白（tdTomato）插入IL-22的基因该荧光蛋白不能被分泌，因此ÿ保持截留在体内产生细胞内。荧光生产者可以然后在组织切片或通过流式细胞仪体外分析来可视化。对于记者的实际施工过程中包括重组工程包含了IL-22基因的细菌人工染色体。然后，该工程化的染色体被引入到小鼠基因组。在不同的小鼠组织中，包括脾，胸腺，淋巴结，淋巴集结，以及肠，通过流式细胞术分析中观察到稳态的IL-22报告表达。结肠炎诱导T细胞（CD4 + CD45RBhigh）转让，并可视化报告基因的表达。阳性T细胞首先出现在肠系膜淋巴结，然后将它们累加远端小肠和结肠组织的固有层中。相比于IL-22 EXPRES使用BAC的策略得到了良好的高保真记者表达锡永，并且它比敲入程序简单。
Introduction
报道基因的细胞类型特异性表达是识别细胞积极地表达于下稳态和摄动态组织中的靶是有用的。它也允许对这些细胞，这保持存活，研究其其它性质的纯化。报告小鼠已被用于在阐明为特定的细胞因子，转录因子，和调节元件的作用机制。先前策略1，2，3在很大程度上依赖于敲记者成在小鼠染色体中，耗时和昂贵的过程中的目标轨迹。因此，对于报告小鼠的生成一个更简单的方法是可取的。
细胞因子是一大类小，分泌的蛋白质/肽调节通过间信号的免疫反应的。白细胞介素22（IL-22）是许多细胞因子报道活动，包括屏障福nction，组织修复和炎症4。虽然IL-22最初被发现作为T细胞产物5，随后的报告证明在人类6和小鼠7和其他类中固有的淋巴细胞8及其在自然杀伤（NK）细胞中表达。尽管IL-22产生细胞的广泛的观察，可视化的IL-22以前需要离体刺激和透的污渍的抗体。因此，新的IL-22的报告小鼠。将研究IL-22的体内平衡和致病过程的功能的非常有用的工具。
在这里，我们开发了一种简化的转基因报告小鼠模型来观察的IL-22产生细胞在体内和体外 。使用BAC重组工程方法9，我们插入的tdTomato cDNA序列与保利的信号碎片进入IL-22 LOC我们和替换外显子1的其它非翻译区，外显子和调控元件没有扰动，因为我们希望尽可能模仿的IL-22的自然调节。记者插入部位扰乱信号序列，从而在生产细胞内的记者的积累，不同于IL-22本身，这是快速分泌。这种新的方法也可以应用到报告小鼠的产生用于其它分泌蛋白。
Protocol
Representative Results
Discussion
IL-22在先天宿主防御和组织重塑中起重要作用。 IL生产-22细胞已经鉴定体外通过细胞内染色。然而，它仍然是难以跟踪的IL-22表达的原位 ，无论是在正常状态还是在炎性病症。这个协议描述来开发的IL-22记者小鼠模型中，这使我们能够本地化记者表达细胞在体内的新方法。报告基因编码TdTomato在扰乱的信号序列的位点插入到IL-22的基因座。在报道这种策略的结果被截留在产生细?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Kelli Czarra and Megan Karwan for animal technical assistance, Kathleen Noer and Roberta Matthai for flow cytometry assistance, and Donna Butcher andMiriam R. Anver for pathology analysis. This project was supported by a grant from the Ely and Edythe Broad Foundation (to Scott Durum) and has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E (MRA).
Materials
| RP23-401E11 BAC | Thermo Fisher Scientific | RPCI23.C | Need gene ID: 50929 |
| NucleoBond BAC 100 | Takara Clontech | 740579 | |
| PCR SuperMix High Fidelity | Thermo Fisher Scientific | 10790020 | |
| PI-SceI | New England Biolabs | R0696S | |
| SpeI | New England Biolabs | R0133S | |
| LB Broth | Thermo Fisher Scientific | 10855-001 | 1L: 10 g SELECT Peptone 140, 5 g SELECT Yeast Extract, 5 g sodium chloride |
| Anti-mouse CD3 | eBioscience | 11-0031 | |
| Anti-mouse CD4 | eBioscience | 17-0041 | |
| Anti-mouse CD45 | Thermo Fisher Scientific | MCD4530 | |
| Anti-mouse CD45RB | eBioscience | 11-0455 | |
| Anti-mouse RFP | Abcam | Ab62341 | |
| HBSS, no calcium, no magnesium, no phenol red | Thermo Fisher Scientific | 14175145 | KCl, KH2PO4, Na2HPO4, NaHCO3, NaCl, D-Glucose |
| Dnase I | Roche | 10104159001 | |
| ACK lysing buffer | Thermo Fisher Scientific | A1049201 | |
| Percoll | GE healthcare life sciences | 17-0891-01 | |
| Collagenase D | Roche | 11088858001 | |
| Dispase II (neutral protease, grade II) | Roche | 4942078001 | |
| IX70 inverted fluorescence microscope | Olympus | Ask for quote | |
| Nikon Eclipse 80i microscope | Nikon | Ask for quote | |
| Dynal shaker | Electron Microscopy Science | 61050-10 | |
| FACSAria | BD Bioscience | Ask for quote | |
| LSRII SORP/flow cytometry | Becton, Dickinson and Company | Ask for quote |
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