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.

1. UV Mutagenesis to Generate Auxotrophic Yeast Strains
<ol>
	<li>Generate survival curves to determine needed doses of UV irradiation
	<ol>
		<li>Prepare YPD (yeast extract peptone dextrose) media and other reagents listed in Table 1 according to standard procedures26 and inoculate single colonies into 5-10 ml of YPD media. Incubate cultures on a roller drum at 30 &#176;C O/N&#160;to saturation for at least 8 hr.</li>
		<li>Determine the cell concentration of O/N cultures using a spectropho...

<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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology, Volume 149: Guide to Yeast Genetics and Molecular Biology. , (1991).</li><li>DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+in+Saccharomyces+cerevisiae+using+CRISPR-Cas+systems.">Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.</a> Nucleic acids research. 41 (7), 4336-4343 (2013).</li><li>Sander, J. D., Joung, J. 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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 border="0" cellpadding="0" cellspacing="0" width="704">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SmartSpec 3000 Spectrophotometer</td>
			<td style="width:107px;">BioRad</td>
			<td style="width:107px;">170-2501</td>
			<td style="width:275px;">Example of spectrophotometer for determining cell concentration and OD600 of yeast cultures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">New Brunswick Roller Drum</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:107px;">M1053-4004</td>
			<td style="width:275px;">Example of roller drum for yeast culture incubation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV Stratalinker 2400</td>
			<td style="width:107px;">Stratagene</td>
			<td style="width:107px;">400075-03</td>
			<td style="width:275px;">Example stratalinker</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Stuart Colony Counter SC6PLUS</td>
			<td style="width:107px;">11983044</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:275px;">Plate stand with magnification records colony count upon sensing pressure from pen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scienceware Colony Counter&#160;</td>
			<td style="width:107px;">F378620002</td>
			<td style="width:107px;">Bel-Art Scienceware</td>
			<td style="width:275px;">Hand held colony counter pen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Replica plating device</td>
			<td style="width:107px;">Fisherbrand</td>
			<td>09-718-1</td>
			<td rowspan="2" style="width:275px;">Example of replica plating stand and pads&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Velveteen squares</td>
			<td style="width:107px;">Fisherbrand</td>
			<td>09-718-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;L shaped sterile cell spreaders&#160;</td>
			<td style="width:107px;">Fisherbrand</td>
			<td style="width:107px;">14665230</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Deoxyribonucleic acid, single stranded from salmon testes</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:107px;">D7656-1ML</td>
			<td style="width:275px;">Example carrier DNA for yeast LiOAc transformation</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Gavage needles</td>
			<td style="width:107px;">Braintree Scientific</td>
			<td style="width:107px;">N-PK&#160;002</td>
			<td style="width:275px;">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 g, 20 gauge or larger straight or curved gavage needles may be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1mL sterile slip-tip disposable tuberculin syringe&#160;</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:107px;">BD 309659</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Blunt forceps such as Electron Microscopy Sciences 7&#34; (178 mm) serrated tip, broad grip forceps</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:107px;">77937-28</td>
			<td style="width:275px;">Example of blunt forceps needed for dissection&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Straight and curved dissection scissors&#160;</td>
			<td style="width:107px;">Electron Microscopy Sciences&#160;</td>
			<td style="width:107px;">72966-02 and 72966-03</td>
			<td style="width:275px;">Examples of scissors needed for dissection&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IMDM</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:107px;">12440053</td>
			<td style="width:275px;"></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...

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

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 ...

The overall goal of these procedures is to develop a strain of probiotic yeast that can be easily transformed to express and deliver heterologous protein to the murine gastrointestinal tract. The main advantage of these techniques is that they allow for rapid generation of oxytrophic strains of probiotic yeast. These mutant yeasts can then be genetically manipulated without relying on antibiotic selection.Oral gavage of mice and harvest of Peyer's patches also allows for verification of mutant yeast adherence to the gastrointestinal immune tissues. These methods will help answer key questions in the field of oral vaccination and facilitate experiments testing probiotics for the delivery of gastrointestinal therapeutics. After preparing YPD medium and growing yeast overnight to saturation, dilute cells one to ten in water in a plastic cuvette.Then, lank a spectrophotometer and determine the optical density of the overnight culture. With 20 milliliters of sterile distilled water, dilute the cells to a concentration of 10 to the seventh cells per milliliter. Then, pour the diluted cells into a sterile plastic petri dish, and with the lid removed, place the plate into a UV crosslinker.Next, expose the cell solution to cumulative incremental doses of UV irradiation, extracting 500 microliter aliquots following each increment to establish a killing curve, and identify the optimal percent survival for screening. Use sterile water to serially dilute the cells at one to ten increments. Then, pellet the cells from each dilution by centrifugation at maximum speed for one minute.Aspirate the supernatant, and resuspend the cells in a 100 microliter volume of sterile water appropriate for plating yeast cells. Then, pipette the full volume of resuspended cells onto plates containing YPD solid medium, and use a sterile spreader to evenly distribute cells across each plate. Plot a survival curve, and determine the dose of UV irradiation to be used for screening according to the text protocol.After exposing yeast to the dose of UV irradiation selected for screening, pellet the full volume of irradiated cells, and use 100 microliters of sterile water to resuspend the pellet. Plate onto minimal medium containing 5-FOA according to the text protocol, in order to select your three minus oxytrophic newtons. Confirm the URA3 minus phenotype by using the tip of a sterile toothpick to collect part of a single colony on the 5-FOA plate, and restreak it onto YPD, 5-FOA, and uracil minus plates.Wrap the plates, and incubate upside down at 30 degrees Celsius for two to four days. Confirm oxytrophic status by verifying growth of colonies on YPD and 5-FOA, but not Uracil minus plates. After confirming the URA-3 minus status of irradiated yeast, set up an overnight filter for lithium acetate transformation, by inoculating 10 milliliters of YPD with a single mutant colony, and incubate in a warm room at 30 degrees Celsius.The following day, use 50 milliliters of fresh, warm YPD to dilute the overnight cultures to an OD600 of 0.16 to 0.2, and incubate the cells on an orbital platform shaker set to 200 RPM, until the culture reaches approximately one times 10 to the seventh cells per mill, usually around four hours. After washing the cells with water and lithium acetate according to the protocol, add 50 microliters of resuspended cells to a transformation mixture of plasmid and carrier DNA. Then, add 300 microliters of PEG, TE, and lithium acetate and vortex.Incubate the preparations at 30 degrees Celsius on an orbital platform shaker at 200 RPM for 30 minutes. Continue with the transformation as described in the text protocol. After preparing yeast cultures according to the text protocol, use a hemocytometer to determine the cell concentration, and adjust to 10 to the ninth cells per milliliter.Pellet the cells by centrifugation at 2500 times G for three minutes. Then, pour off the supernatant and resuspend the cells by adding the appropriate volume of sterile water, and gently pipetting up and down. Fix an appropriate gauge gavage needle onto a one milliliter sterile syringe, and load the yeast sample, eliminating any air bubbles.Load an additional syringe with sterile water to gavage control mice. Using the non-dominant hand, pick up the mouse to be gavaged and, with the index finger and thumb, tightly grasp the skin around the neck. Tuck the tail under the small finger.Be sure the grip is secure and prohibits the mouse from moving its head. Next, gently insert the gavage needle into the mouse's esophagus by angling the needle along the roof of the mouth and back of the throat, keeping slightly to the left of center. Wait for the mouse to swallow the bulb of the needle.If resistance is felt or the mouse gasps, gently remove the needle, and try again to find the esophagus. After the mouse has swallowed the bulb of the gavage needle, gently depress the syringe plunger to administer the yeast directly into the mouse stomach. Gently remove the gavage needle from the mouse stomach and esophagus, and return the mouse to the cage.Check that the mouse is breathing and moving normally to ensure the gavage was performed properly. After sacrificing the mouse according to the text protocol, position it with the abdomen fully exposed and use 70%ethanol to spray the abdominal area. With scissors, make a transverse incision through the fur and skin.Then manually pry the incision open to further expose the peritoneum, the thin serosal lining that covers the abdominal organs. Gently lift the peritoneum, and make a transverse incision to expose the intestines. Then, use blunt forceps to carefully tease the small intestine away from the mesenteric arteries, fat, and other tissues.Expose the small intestine from the stomach in the upper left quadrant of the mouse abdomen to the cecum, the large pocket of intestinal tissue at the start of the large intestine. Next, isolate Peyer's patches by looking for one to two millimeter roughly circular patches of opaque tissue along the small intestine. Then, using curved dissection scissors, cut away the dome of the Peyer's patch, leaving margins to ensure that none of the surrounding tissue is collected.Transfer the dissected Peyer's patches into complete IMDM. Then, pour the solution with suspended Peyer's patches onto a 40 micrometer cell strainer. After washing the Peyer's patches with complete IMDM, use a plunger from a one milliliter syringe to gently break up the tissue, and collect the cells in a 50 milliliter tube.Pellet the strained cells at 1800 RPM for seven minutes. Then, plate and analyze the cells according to the text protocol. This figure shows a plating from a serial dilution of irradiated S Boulardii cells.The serial dilution ensures that the CF use could be enumerated at each UV dose. From these images of yeast plates, the consistent growth of mutant colonies on YPD and 5-FOA, but not Uracil minus plates, indicates a Uracil minus oxytrophic phenotype. This graph illustrates the transformation efficiency for wild type S Boulardii, compared to S Cerevisiae, using both lithium acetate and electroporation techniques.Although lithium acetate transformation is very efficient for S Cerevisiae, transformation efficiency for S Boulardii is greatly improved using electroporation. In these Brightfield and Fluorescence images, S Cerevisiae transformed with a URA3 plasmid encoating GFE demonstrates a functional expression of heterologous protein. In this figure, an example of viable CF use detected from Peyer's patches after animal dissection is shown.A typical yield of CF use recovered per mouse is less than 10. You should now have a good understanding of how to generate oxytrophic mutant strains of probiotic yeast, using UV mutogenesis. We have also described how to test the ability of these yeasts to express heterologous protein and adhere to Peyer's patches of the mouse small intestine.Following these procedures, other methods, such as ELISA and fluorescence microscopy can be used to evaluate proper folding and function of heterologous proteins expressed.

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JoVE Journal

Immunology and Infection

12.2K 조회수.  Emory University School of Medicine. The overall goal of these procedures is to develop a strain of probiotic yeast that can be easily transformed to express and deliver heterologous protein to the murine gastrointestinal tract. The main advantage of these techniques is that they allow for rapid generation of oxytrophic strains of probiotic yeast. These mutant yeasts can then be genetically manipulated without relying on antibiotic selection.Oral gavage of mice and harvest of Peyer's patches also allows for verification o...