Name: visualization of il-22-expressing lymphocytes using reporter mice
Uploaded: 2017-01-25T12:30:00
Description: Watch this Scientific Journal Video about Visualization of IL-22-expressing Lymphocytes Using Reporter Mice at JoVE.com

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).

All animals received proper care in accordance with the experimental procedures outlined in the 2011 Guide for Care and Use of Laboratory Animals Committee of Frederick National Laboratory for Cancer Research.
1. Generation of IL-22-tdTomato Reporter Mice by BAC Recombineering
NOTE: The mice should be unconscious and do not move in response to a noxious stimulus. Sterilize the surgical area with 70% ethanol and sterilize all surgical tools using a glass bead sterilize...

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IL-22 plays an essential role in innate host defense and tissue remodeling. IL-22-producing cells have been identified ex vivo by intracellular staining. However, it still remains difficult to track IL-22 expression in situ, either in the normal state or in inflammatory conditions. This protocol describes a novel method to develop an IL-22 reporter mouse model, which enables us to localize the reporter-expressing cells in vivo. The reporter gene encoding TdTomato was inserted into the IL-22 loc...

Cell type-specific expression of reporter genes is useful to identify cells actively expressing the target in tissues under homeostatic and perturbed states. It also allows for the purification of these cells, which remain viable, to study their other properties. Reporter mice have been utilized in elucidating the mechanism of action for specific cytokines, transcription factors, and regulatory elements. Previous strategies1,2,3 have largely relied on knocking the reporter into the target locus in the mouse chromosome, a time-consuming and costly procedure. Thus, a simpler method for the generation of reporter mice is desirable.
Cytokines are a broad class of small, secreted proteins/peptides that regulate immune responses through intercellular signaling. Interleukin 22 (IL-22) is a cytokine with many reported activities, including barrier function, tissue repair, and inflammation4. Although IL-22 was initially discovered as a T-cell product5, subsequent reports demonstrated its expression in natural killer (NK) cells in humans6 and mice7 and in other classes of innate lymphocytes8. Despite extensive observation of IL-22-producing cells, visualization of IL-22 previously required ex vivo stimulation and permeabilization to stains with antibodies. Therefore, novel IL-22 reporter mice would be a very useful tool to investigate the function of IL-22 in homeostatic and pathogenic processes.
Here, we developed a simplified transgenic reporter mouse model to observe the IL-22-producing cells in vivo and in vitro. Using a BAC recombineering method9, we inserted the tdTomato cDNA sequence with Poly A signal fragments into the IL-22 locus and replaced exon 1. The other untranslated regions, exons, and regulatory elements were not perturbed, since we would like to mimic the natural regulation of IL-22 as much as possible. The site of reporter insertion disrupts the signal sequence, resulting in the accumulation of the reporter inside the producing cells, unlike IL-22 itself, which is rapidly secreted. This new method can also be applied to the generation of reporter mice for other secreted proteins.

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			<td height="21" style="height:21px;width:164px;">RP23-401E11 BAC</td>
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			<td style="width:151px;">Abcam</td>
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			<td height="61" style="height:61px;width:164px;">HBSS, no calcium, no magnesium, no phenol red</td>
			<td style="width:151px;">Thermo Fisher Scientific</td>
			<td style="width:116px;">14175145</td>
			<td style="width:345px;">KCl, KH2PO4, Na2HPO4, NaHCO3, NaCl, D-Glucose</td>
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visualization of il-22-expressing lymphocytes using reporter mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

A murine IL-22 reporter transgene was created using recombineering to modify a bacterial artificial chromosome carrying the IL-22 locus. Figure 1 shows a diagram of pBACe3.6 vector containing the sacBII gene, a positive-selection marker, and chloramphenicol antibiotic resistance gene11. After introducing tdTomato into exon 1, the signal peptide sequence was disrupted, as shown in Figure 2. Thus, the tdTomato reporter was t...

Watch this Scientific Journal Video about Visualization of IL-22-expressing Lymphocytes Using Reporter Mice at JoVE.com

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

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.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

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Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Intravital Imaging of Neutrophil Priming Using IL-1ÃƒÅ½Ã‚Â² Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...