The authors acknowledge funding through the Children&#39;s Center for Immunology and Vaccines and an NIH New Innovator Award (1DP2AI112242-01) awarded to Tracey J. Lamb. The authors also thank Natalya P. Degtyareva for the generous contribution of rad1 S. cerevisiae.

<ol>
	<li>Edwards-Ingram, L., Gitsham, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+and+physiological+characterization+of+Saccharomyces+boulardii,+the+probiotic+strain+of+Saccharomyces+cerevisiae.">Genotypic and physiological characterization of Saccharomyces boulardii, the probiotic strain of Saccharomyces cerevisiae.</a> Applied and environmental microbiology. 73 (8), 2458-2467 (2007).</li><li>Hudson, L. E., Fasken, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Heterologous+Protein+Expression+by+Genetically+Engineered+Probiotic+Yeast+Saccharomyces+boulardii.">Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii.</a> PloS one. 9 (11), 1-12 (2014).</li><li>Guthrie, C., Fink, G. 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The authors have nothing to disclose.

Together, the protocols herein describe the essential steps necessary for the development and testing of auxotrophic probiotic yeast strains for delivery of heterologous therapeutic protein to the intestine. This manipulation and testing of recombinant probiotic yeast requires techniques and resources with which any individual laboratory may not currently be familiar. Thus, although numerous previous studies have described the above protocols for multiple yeast and mouse strains, these methods have not to the authors&#39...

Probiotic microorganisms are an intriguing potential means of efficiently and economically delivering heterologous proteins to the gastrointestinal tract. These organisms are capable of surviving passage through the gastrointestinal tract yet do not colonize it1, enabling controlled dosing and limiting exposure to the drug expressed. Furthermore, the ability to easily engineer these organisms to produce heterologous protein on a large scale renders them an economical alternative to synthetic delivery particles. However, development of such an approach, as recently demonstrated using an auxotrophic strain of the probiotic yeast Saccharomyces boulardii2, requires knowledge of laboratory techniques not traditionally combined within a given study, ranging from yeast and molecular biology to animal handling techniques and immunological methods. Thus although the individual procedures described herein are not in themselves novel laboratory protocols, the goal of this manuscript is to present a unified introduction to techniques needed for experimental testing of probiotic yeast as drug delivery vehicles to the murine gastrointestinal tract. Provided is a compilation of essential protocols for: 1) generation of auxotrophic mutant strains of yeast that can easily be genetically manipulated; 2) transformation of yeast cultures to express heterologous protein; 3) administration of recombinant yeast to the intestine via oral gavage; and 4) recovery of viable recombinant probiotic yeast from the murine intestine and assessment of their heterologous protein expression.
First, although numerous positive and negative selection methods exist for the manipulation of yeast species, negative selection such as through the use of auxotrophic markers increases both the efficiency and ease with which yeast can be transformed and selected. Positive selection of transformants using antibiotics, in contrast, significantly increases the cost of yeast manipulation. Furthermore, selection of yeast on antibiotic-containing solid media can allow for increased growth of untransformed background colonies relative to selection of auxotrophic yeast on synthetic drop out solid media (unpublished observations). Auxotrophic yeast is strains which lack enzymes critical for the synthesis of essential amino acids or uracil. Such yeast can grow only if supplemented with the missing metabolite or metabolic gene, thus enabling negative selection when yeast is plated onto synthetic drop out media that lacks the essential metabolite. Many commonly used Saccharomyces cerevisiae laboratory strains are in fact already auxotrophic mutants3. Industrial, clinical, and probiotic yeast strains, however, are typically prototrophic with the ability to synthesize all required nutrients. To enable more efficient genetic manipulation of such yeast, auxotrophic genes can be selectively targeted to generate strains that can be selected without antibiotics. Specific targeting of auxotrophic marker genes can be achieved through PCR-mediated gene disruption relying on homologous recombination or more recently through CRISPR/Cas9 targeting4-6. Alternatively, UV mutagenesis can quickly generate auxotrophic mutants even in yeast strains for which transformation with multiple plasmids is technically difficult7. While PCR targeting and CRISPR/Cas9 have been described extensively elsewhere, presented in part one of this manuscript is a detailed protocol describing a UV mutagenesis approach to create auxotrophic strains that will allow for negative selection rather than positive antibiotic selection of yeast transformants.
The next necessary step in the use of such auxotrophic strains for oral delivery of heterologous protein is yeast transformation with plasmid DNA. Since the first successful transformation of yeast spheroplasts reported for Saccharomyces cerevisiae in 19788, numerous modifications have been characterized to increase the efficiency and ease with which yeast species can be genetically modified. Use of electroporation for the successful transformation of DNA into S. cerevisiae was first described in 19859 and has since been improved via the addition of 1 M sorbitol incubation to osmotically support cells10. Electroporation efficiency has furthermore been shown to depend on the yeast species and strain, cell number and phase of growth, electroporation volume, field strength, and specific buffers11. Lithium acetate (LiOAc) transformation, originally described by Ito et al.12, is among the most commonly used transformation protocols as it requires no special equipment. Additional analyses showed that the efficiency of LiOAc yeast transformation greatly increases when cells are collected in mid-log phase of growth and are heat shocked in the presence of polyethylene glycol (PEG) and DNA at 42 &#176;C12. Incubation of whole intact yeast with PEG is essential for efficient transformation, possibly through improving attachment of DNA to the cell membrane as well as via other effects on the membrane13. Lithium itself also increases the permeability of intact cells14. Although most laboratory S. cerevisiae strains can easily be transformed using LiOAc transformation3, other yeast species may be more efficiently transformed using alternative protocols. Pichia pastoris, for example, is most efficiently transformed via electroporation rather than LiOAc transformation13. It is crucial, therefore, to test multiple methods of transformation and to optimize incubation periods and reagent concentrations when attempting to genetically modify an uncharacterized yeast strain. This manuscript thus describes both LiOAc transformation and electroporation as techniques for the transformation of auxotrophic mutant and wild type S. boulardii. Interested readers are directed to recent reviews for thorough descriptions of the evolution of yeast transformation, alternative protocols, and further discussions of possible mechanisms of action13,15. Transformation of yeast with plasmid encoding an easily detectable protein is furthermore essential for downstream testing in order to ensure proper expression and function of heterologous protein. Myriad different proteins may be selected depending on the ultimate purpose of the therapeutic study and the antibodies available for protein detection by immunoblotting, ELISA, and other techniques. Protocols for these techniques have been thoroughly described elsewhere16,17, and can be used to determine levels of heterologous protein production from transformed yeast by comparison to standard curves. For purposes of demonstration and to show successful production of a very commonly used protein in yeast biology, this manuscript presents transformation with plasmid encoding green fluorescent protein (GFP), which allows for subsequent detection using fluorescence microscopy.
Equally important to the production of probiotic organisms that express heterologous protein is the proper administration and detection of these microorganisms within gastrointestinal tissues, as described in parts three and four. Administration of recombinant yeast via oral gavage allows for delivery of controlled quantities of yeast directly into the stomach, from which C57BL/6 mice are naturally incapable of vomiting18. However, improper animal handling and gavage can lead to esophageal damage and perforation, gastric perforation, tracheal administration, and aspiration pneumonia19,20. Poor technique and inexperience can furthermore increase variability in murine immune responses and experimental results, which have been attributed to animal stress upon oral gavage21,22. Practice in the proper technique can thus not only attenuate animal discomfort, but can also increase precision of experimental results. This manuscript describes and demonstrates animal handling and oral gavage for the administration of controlled doses of recombinant yeast.
Finally, it is vital to confirm successful delivery of recombinant yeast by analyzing lymphoid tissues for the presence of yeast and heterologous protein. The gastrointestinal immune tissues which can most easily and predictably be examined for the presence of yeast are the Peyer&#39;s patches. Peyer&#39;s patches are secondary lymphoid organs along the small intestine that are key sites of mucosal immune response induction23. Antigens from the lumen are transferred transcellularly through microfold (M) cells in the epithelium and are released into the Peyer&#39;s patches, thus exposing enclosed antigen presenting cells to intestinal luminal contents. Although particle uptake across the intestinal epithelium can also be achieved by goblet cells, these cells have been shown to only take up particles less than 0.02 &#181;m in diameter24. Transepithelial dendrites extended from CD103+ dendritic cells (DC) also take up small particles from the intestinal lumen25; however, there are currently no reports demonstrating that CD103+ DCs take up particles larger than bacteria. Thus, intact probiotic yeast, of average size between 3-6 &#181;m in diameter, are most likely to be taken up by M cells and transferred to the Peyer&#39;s patches. Described here is a protocol for collection and screening of Peyer&#39;s patches for viable recombinant yeast, although this procedure can also be easily adapted for evaluating uptake of probiotic bacteria.
In summary, assessing recombinant probiotic yeast for the delivery of therapeutic proteins to the intestine requires proficiency in laboratory techniques spanning molecular biology to animal handling and immunology. Presented here are protocols for 1) the generation and screening of auxotrophic yeast strains which can be easily negatively selected without antibiotics, 2) alternative protocols to transform yeast and enable expression of heterologous protein, 3) demonstrations of proper animal handling techniques and oral gavage for intragastric delivery of recombinant yeast, and 4) protocols for Peyer&#39;s patch dissection and screening for viable recombinant yeast and functional heterologous protein. Combined, these protocols will allow for the generation and testing of a probiotic yeast strain capable of delivering heterologous therapeutic protein to the gastrointestinal tract.

<table><tbody><tr><td>SmartSpec 3000 Spectrophotometer</td><td>BioRad</td><td>170-2501</td><td>Example of spectrophotometer for determining cell concentration and OD&lt;sub&gt;600&lt;/sub&gt; of yeast cultures</td></tr><tr><td>New Brunswick Roller Drum</td><td>Eppendorf</td><td>M1053-4004</td><td>Example of roller drum for yeast culture incubation</td></tr><tr><td>UV Stratalinker 2400</td><td>Stratagene</td><td>400075-03</td><td>Example stratalinker</td></tr><tr><td>Stuart Colony Counter SC6PLUS</td><td>11983044</td><td>Fisher Scientific</td><td>Plate stand with magnification records colony count upon sensing pressure from pen</td></tr><tr><td>Scienceware Colony Counter&amp;nbsp;</td><td>F378620002</td><td>Bel-Art Scienceware</td><td>Hand held colony counter pen</td></tr><tr><td>Replica plating device</td><td>Fisherbrand</td><td>09-718-1</td><td>Example of replica plating stand and pads&amp;nbsp;</td></tr><tr><td>Velveteen squares</td><td>Fisherbrand</td><td>09-718-2</td><td></td></tr><tr><td>L shaped sterile cell spreaders&amp;nbsp;</td><td>Fisherbrand</td><td>14665230</td><td></td></tr><tr><td>Deoxyribonucleic acid, single stranded from salmon testes</td><td>Sigma-Aldrich</td><td>D7656-1ML</td><td>Example carrier DNA for yeast LiOAc transformation</td></tr><tr><td>Gavage needles</td><td>Braintree Scientific</td><td>N-PK&amp;nbsp;002</td><td>For mice 15-20 g, the suggested needle is a 22 gauge (1.25 mm ball), 1 in long, straight reusable gavage needle. For mice weighing greater than 20&amp;nbsp;g, 20 gauge or larger straight or curved gavage needles may be used</td></tr><tr><td>1 ml sterile slip-tip disposable tuberculin syringe</td><td>Becton Dickinson</td><td>BD 309659</td><td></td></tr><tr><td>Blunt forceps such as Electron Microscopy Sciences 7&quot; (178 mm) serrated tip, broad grip forceps</td><td>Electron Microscopy Sciences</td><td>77937-28</td><td>Example of blunt forceps needed for dissection&amp;nbsp;</td></tr><tr><td>Straight and curved dissection scissors</td><td>Electron Microscopy Sciences</td><td>72966-02 and 72966-03</td><td>Examples of scissors needed for dissection</td></tr><tr><td>IMDM</td><td>Life technologies</td><td>12440053</td><td></td></tr></tbody></table>

transformation of probiotic yeast and their recovery from gastrointestinal immune tissues following oral gavage in mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Generation of a survival curve following UV irradiation requires plating of diluted yeast cells such that distinct colony forming units (CFU) are able to form. Each 500 &#181;l sample collected as described above contains approximately 5 x 106 cells; however, greater than 100 colonies per plate are difficult to accurately distinguish. Plating undiluted sample as well as serial 1:10 dilutions of irradiated cells thus ensures that CFU can be enumerated at each UV dose, as demonst...

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Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

transformation-probiotic-yeast-their-recovery-from-gastrointestinal

Research

JoVE Journal

Immunology and Infection

12.3K Views.  Emory University School of Medicine. Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Video: Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice.

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

Medicine

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting cells. When the skin encounters invading pathogens, different cutaneous DCs initiate a distinct T cell immune response to protect the body. Among the invading pathogens, fungal infection specifically drives a protective interleukin-17-producing Th17 immune response. A protocol was developed to efficiently differentiate Th17 cells by intradermal Candida albicans infection to investigate a subset of cutaneous DCs responsible for inducing Th17 immunity. Flow cytometry and gene expression analyses revealed a prominent induction of Th17 immune response in skin-draining lymph nodes and infected skin. Using diphtheria toxin-induced DC subset-depleting mouse strains, CD301b+ dermal DCs were found to be responsible for mounting optimal Th17 differentiation in this model. Thus, this protocol provides a valuable method to study in vivo function of differential subsets of cutaneous DCs to determine Th17 immunity against deep skin fungal infection.

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

Here, we demonstrate the in vivo function of cutaneous dendritic cell subsets in Th17 immunity of deep dermal Candida albicans infection.

The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting ...

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted considerable attention. Accumulating evidence suggests that dysbiotic microbiota plays an active role in triggering or worsening the inflammatory process in IBD and that fecal microbiota transplantation (FMT) is an attractive therapeutic strategy since transferring a healthy microbiota to IBD patient could restore the appropriate host-microbiota communication. However, the molecular mechanisms are unclear, and the efficacy of FMT has not been very well established. Thus, further studies in animal models of IBD are necessary. In this method, we applied FMT from wild-type C57BL/6J mice to IL-10 deficient mice, a widely used mouse model of colitis. The study elaborates on collecting fecal pellets from the donor mice, making the fecal solution/suspension, administering the fecal solution, and monitoring the disease. We found that FMT significantly mitigated the cardiac impairment in IL-10 knockout mice, underlining its therapeutic potential for IBD management.

The interaction of genetic susceptibility, mucosal immunity, and intestinal microecological environment is involved in the pathogenesis of inflammatory bowel disease (IBD). In this study, we applied fecal microbiota transplantation to IL-10 deficient mice and investigated its impact on colonic inflammation and heart function.

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted ...

Murine Fecal Isolation and Microbiota Transplantation

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal microbiota transplantation (FMT) is a valuable approach to delineating a direct role of the total microbiota or isolated species in disease pathophysiology. It is a safe treatment option for patients with recurrent Clostridium difficile infection. Preclinical studies demonstrate that manipulating the gut microbiota is a useful tool to study the mechanistic link between dysbiosis and disease. Fecal microbiota transplantation may help elucidate novel gut microbiota-targeted therapeutics for the management and treatment of cardiometabolic disease. Despite a high success rate in rodents, there remains translational changes associated with the transplantation. The goal here is to provide guidance in studying the effects of gut microbiome in experimental cardiovascular disease. In this study, a detailed protocol for the collection, handling, processing, and transplantation of fecal microbiota in murine studies is described. The collection and processing steps are described for both human and rodent donors. Lastly, we describe using a combination of the Swiss-rolling and immunostaining techniques to assess gut-specific morphology and integrity changes in cardiovascular disease and related gut microbiota mechanisms.

The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract.

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal ...

Preparation and Maintenance of Bioexclusion IsoPositive Cage Experiment for Human Fecal Transplantation into Germ-Free Mice

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of the specific role of individuals, defined or complex groups of microorganisms in host response. Traditionally bred and reared in flexible-film or semi-rigid isolators, germ-free mouse husbandry and experimental manipulation are costly and require numerous trained staff and a large space footprint in animal housing facilities. The IsoPositive caging system allows for experimental manipulation of germ-free mice in individual, hermetically-sealed, positive-pressure isolator cages (isocages), reducing cost and enabling greater flexibility in experimental manipulations. 
Here, a protocol is described for transferring germ-free mice from breeding isolators to isocages and subsequent fecal transfer from human donor stool into mice to create stable long-term gut "humanized" mice for future studies. The materials and preparation needed for the utilization of the isocage system are described, including the use of chlorine-dioxide sterilant chemical sterilant to clean cages, supplies, equipment, and personal protective equipment. The methods for confirming the germ-free status of transferred mice and how to determine contamination in the caging system are discussed. The procedure for husbandry, including bedding, food, and water supply, is further discussed. The protocol for human fecal slurry preparation and gavage into germ-free mice to create gut "humanized" mice, along with stool collection to monitor the microbial community composition of these mice, are described. An experiment illustrates that two weeks post-human fecal transplant allows for stable colonization of donor microbiota in the murine hosts, enabling subsequent experimental usage. Furthermore, the collection of humanized mouse feces in viability preservation media, enabling use in further functional experiments, is described. Overall, these methods allow for the safe and effective establishment of humanized mouse communities in experimental gnotobiotic cages for further manipulation.

This protocol describes the best practices for germ-free mouse transfer to and housing in experimental single-cage isolators (isocages) while maintaining sterile conditions. Methods for fecal transplant into germ-free mice and the collection of viable bacteria from these gut "humanized" mice for further applications are discussed.

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

Fecal Microbiota Transplantation and Detection of Prevalence of IgA-Coated Bacteria in the Gut

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the biological function of intestinal bacteria as a whole or at the species level. Several different FMT methods have been published. Here, we present an FMT protocol that successfully depletes gut microbiota in a matter of days, followed by transplantation of fecal microbiota from fresh or frozen donor intestinal contents to conventional mice. Real time-PCR is applied to test the efficacy of bacterial depletion. Sequencing of the 16S ribosomal RNA (rRNA) is then applied to test the relative abundance and identity of gut microbiota in recipient mice. We also present a flow cytometry-based detection method of immunoglobulin A (IgA)-coated bacteria in the gut.

We established an efficient way to deplete intestinal bacteria in three days, and subsequently transplant fecal microbiota via gavage of fecal fluid prepared from fresh or frozen intestinal contents in mice. We also present an optimized method to detect the frequency of IgA-coated bacteria in the gut.

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the ...

Biology

Evaluating the Effects of Fecal Microbiota Transplantation in an Interleukin-10 Deficient Mouse

Source: Xiao, Y. et al., <a href="https://app.jove.com/v/63350">Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates a procedure of fecal microbiota transplantation in an interleukin-10 deficient mouse with colon inflammation. Colonized fecal microbes in the gut release secondary metabolites, eliciting anti-inflammatory cytokine release and reducing colon inflammation.

Ocena skutków przeszczepu mikrobioty kałowej u myszy z niedoborem interleukiny-10

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

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Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4⁺CD45RB^high T Cells into Immunodeficient Mice

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

Murine Fecal Isolation and Microbiota Transplantation

Preparation and Maintenance of Bioexclusion IsoPositive Cage Experiment for Human Fecal Transplantation into Germ-Free Mice

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4⁺ T Cells to Immunodeficient Mice

Fecal Microbiota Transplantation and Detection of Prevalence of IgA-Coated Bacteria in the Gut

Ocena skutków przeszczepu mikrobioty kałowej u myszy z niedoborem interleukiny-10