Name: transformation of probiotic yeast and their recovery from gastrointestinal immune tissues following oral gavage in mice
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Description: Watch this Scientific Journal Video about Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice at JoVE.com

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.

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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 ...

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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

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Probiotic Studies in Neonatal Mice Using Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

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 ...

Exploring Gut Phage-Bacteria Dynamics in Murine Models Using T4 Phage

T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. There has been renewed interest in phage research in the last decade, in part due to the host-specific killing capabilities of lytic phages, which offer a promising tool to counter the increasing threat of antimicrobial resistant bacteria. Furthermore, recent studies demonstrating that phages adhere to intestinal mucus suggest they may have a protective role in preventing bacterial invasion into the underlying epithelium. Importantly, like bacterial microbiomes, disrupted phageomes have been associated with worsened outcomes in diseases such as inflammatory bowel disease. Previous studies have demonstrated that phages can modulate the microbiome of animals and humans through fecal filtrate transplants, benefiting the host&#39;s health. With this recent wave of research comes the necessity to establish and standardize protocols for studying phages in the context of the gut microbiome. This protocol provides a set of procedures to study isolated T4 phages and their bacterial host, Escherichia coli,&#160;in the context of the murine gastrointestinal tract. The methods described here outline&#160;how to start from a phage lysate, administer it to mice and assess effects on bacterial host and phage levels.&#160;This protocol can be modified and applied to other phage-bacterial pairs and provides a starting point for studying host-phage dynamics in vivo.

Author Spotlight: Investigating Bacteriophage-Induced Immune Responses in Gnotobiotic Mice

Bacteriophages (phages), viruses that infect bacteria, are an integral component of the gut microbiome. Though these symbiotic inhabitants drive bacterial fitness and population dynamics, little is understood about how they impact gut homeostasis and disease. This protocol studies isolated T4 phages within a mouse model, adaptable to other phage-bacterial pairs.

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across ...

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 ...

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

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Gut microbiota dysbiosis is thought to exert a role in the progression of colitis. However, the precise standards for probiotic administration in alleviating colitis remain undefined. Most analysis methods rely on limited diversity and abundance of gut microorganisms. Therefore, observational studies cannot establish causation. In this study, we applied antibiotic-induced pseudo-germ-free mice to investigate the role of gut microbiota in regulating the probiotic effects of Bacillus&#160;cereus (B. cereus)&#160;on dextran sulfate sodium (DSS)-induced colitis in mice. This process allows for evaluating the bidirectional regulating effect of B. cereus&#160;supplementation on health and provides stable and reproducible results. Here, the detailed protocols for B. cereus cultivation, gavage operation, stool collection, and antibiotic clearance treatment on colitis mice are provided. The optimization methods are also applicable for other chronic inflammatory-associated disorders. The results showed that B. cereus administration decreased body weight loss, colon length shortening, disease activity index, and histopathological scores. However, treatment with antibiotics suppressed the positive effect of B. cereus on colitis. These results indicate that gut microbiota are needed for the alleviating effects of B. cereus on colitis. Therefore, exploring the beneficial effects of probiotics in this research is a promising approach for developing novel treatment strategies for alleviating the symptoms of chronic inflammatory-associated disorders.

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Here, we present a protocol to establish the antibiotic-induced pseudo-germ-free dextran sulfate sodium-induced colitis mouse model to investigate the role of gut microbiota in regulating the positive effects of Bacillus cereus on colitis.

Gut microbiota dysbiosis is thought to exert a role in the progression of colitis. However, the precise standards for probiotic administration in ...

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.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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Immunotherapy

Model Development and Disease Study

Oral Delivery of Transformed Yeast Cells into the Mouse Intestinal Immune System

Source: Hudson, L. E. et al., <a href="https://app.jove.com/v/53453">Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice.</a>&#160;J. Vis. Exp. (2016)
This video showcases the oral administration of transformed yeast cells into a mouse model. The transformed yeast cells are orally administered using a gavage technique, reaching the mouse&#39;s stomach. Later, the cells migrate to the small intestine, where microfold cells engulf them and facilitate their transfer to Peyer&#39;s patches and gain access to the intestinal immune system.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. UV ...

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

Summary

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Chapters in this video

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

Probiotic Studies in Neonatal Mice Using Gavage

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

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

T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo

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

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

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

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

Oral Delivery of Transformed Yeast Cells into the Mouse Intestinal Immune System