Source: Hudson, L. E. et al., Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice. 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's stomach. Later, the cells migrate to the small intestine, where microfold cells engulf them and facilitate their transfer to Peyer'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 Mutagenesis to Generate Auxotrophic Yeast Strains
2. Yeast Transformation
Table 1. Reagents List. Described are the reagents needed for making each of the solutions, yeast media and plates, and transformation buffers used for the protocols in this manuscript.
Solutions | Yeast Media and Plates | Transformation Reagents |
Polyethylene glycol (PEG) 50%: | YPD: | TE/LiOAc: |
250 g PEG 3350 | 20 g peptone | 50 ml 10x TE |
500 ml sterile water | 20 g dextrose | 50 ml 10x (1M) LiOAc |
Filter sterilized | 10 g yeast extract | 400 ml sterile water |
1 L water | Filter sterilized | |
Autoclave | ||
TE 10x: | YPD plates: | PEG/TE/LiOAc: |
100 mM Tris | 20 g peptone | 400 ml 50% PEG |
10 mM EDTA | 20 g dextrose | 50 ml 10x TE |
pH to 7.5 and filter sterilize | 20 g agar | 50 ml 10x (1M) LiOAc |
10 g yeast extract | ||
1 L water | ||
Autoclave | ||
20% glucose: | Uracil– selective media | Carrier DNA (SS DNA): |
200 g dextrose | 2 g amino acid mix lacking uracil | Store at -20 °C and prior to use heat for 1-2 min at 100 °C to melt strands and store on ice |
1 L water | 6.7 g yeast nitrogen base without amino acids | |
Filter sterilized | 1 L water | |
Sterilize by autoclaving or sterile filtering | ||
Add 20% glucose 1:10 before use | ||
50% glycerol: | Uracil– plates: | Electroporation buffer: |
500 ml glycerol | In a 250 ml flask: | 1 M Sorbitol |
500 ml water | 2 g amino acid mix lacking uracil | 1 mM CaCl2 |
Autoclave | 6.7 g yeast nitrogen base without amino acids | Fill with distilled water |
150 ml water | Autoclave and store at 4 °C | |
In a 2 L flask: | ||
20 g agar | ||
750 ml water | ||
Autoclave flasks separately, then mix together with 100 ml 20% glucose | ||
Complete IMDM | 5-FOA+ plates: | LiOAc/DTT |
500 ml Iscove's Modified Dulbecco's Media | Autoclave in a 2 L flask: | 0.1 M LiOAc |
5 ml penicillin streptomycin glutamine 100x | 20 g agar | 10 mM DTT |
500 μl 2-mercaptoethanol | 750 ml water | |
10% heat inactivated fetal bovine serum | Mix: | |
2.5 ml sodium pyruvate 100 mM | 6.7 g yeast nitrogen base without amino acids | |
2 g amino acid mix without uracil | ||
150 ml warm water | ||
When cool, add: | ||
0.05 g uracil powder | ||
1 g 5-FOA | ||
Stir and filter sterilize | ||
Add to autoclaved agar solution | ||
Mix with 100 ml 20% glucose |
Figure 1. Yeast colonies grown on YPD media. Example YPD plate showing viable colony forming units (CFU) of probiotic yeast after UV irradiation. Cells were serially diluted such that individual CFU can be distinguished and counted.
Figure 2. Survival curve for diploid probiotic yeast. Number of viable S. boulardii CFU as a percent of total plated cells was plotted for each µJ dose of UV irradiation (solid line). The vertical red line indicates the µJ UV dose corresponding to 50% survival of this yeast strain. A rad1 S. cerevisiae mutant, which cannot repair damage from UV mutagenesis, is shown as a control (dashed line).
Figure 3. Confirmation of ura3– phenotype of UV irradiated cells on YPD, uracil–, and 5-FOA plates. Cells from individual UV mutant colonies were collected using the tip of a sterile toothpick and gently streaked across YPD, uracil–, and 5-FOA plates. Cells were first streaked in two perpendicular crossing lines, then a new toothpick was used to pass through the second line and continue spreading cells until individual cells separate. A true ura3– mutant (mut) grows on YPD media and in the presence of 5-FOA, but not in the absence of uracil. Control ura3– S. cerevisiae (ura3–) and URA3+ S. boulardii (URA3+) are shown for comparison and to confirm proper preparation of yeast media.
Figure 4. Transformation Efficiency of Saccharomyces strains. Wild type S. boulardii (S.b.) and a laboratory S. cerevisiae strain (S.c.) were transformed using the described LiOAc (LiOAc) and electroporation (Electro) protocols. Results are plotted as mean CFU obtained per µg of plasmid encoding a kanamycin resistance marker. Bars show the mean of duplicate experiments with error bars depicting the standard error of the mean.
Figure 5. Proper handling of a C57BL/6 mouse for oral gavage. The mouse is held tightly in the non-dominant hand with the tail tucked under the small finger so that no movement is possible (A). The gavage needle is inserted into the pharynx along the roof of the mouth. The mouse is allowed to swallow the bulb of the gavage needle, allowing the solution to then enter the stomach as the plunger is depressed (B).
The authors have nothing to disclose.
SmartSpec 3000 Spectrophotometer | BioRad | 170-2501 | Example of spectrophotometer for determining cell concentration and OD600 of yeast cultures |
New Brunswick Roller Drum | Eppendorf | M1053-4004 | Example of roller drum for yeast culture incubation |
UV Stratalinker 2400 | Stratagene | 400075-03 | Example stratalinker |
Stuart Colony Counter SC6PLUS | 11983044 | Fisher Scientific | Plate stand with magnification records colony count upon sensing pressure from pen |
Scienceware Colony Counter | F378620002 | Bel-Art Scienceware | Hand held colony counter pen |
Replica plating device | Fisherbrand | 09-718-1 | Example of replica plating stand and pads |
Velveteen squares | Fisherbrand | 09-718-2 | |
L shaped sterile cell spreaders | Fisherbrand | 14665230 | |
Deoxyribonucleic acid, single stranded from salmon testes | Sigma-Aldrich | D7656-1ML | Example carrier DNA for yeast LiOAc transformation |
Gavage needles | Braintree Scientific | N-PK 002 | 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 |
1mL sterile slip-tip disposable tuberculin syringe | Becton Dickinson | BD 309659 | |
Blunt forceps such as Electron Microscopy Sciences 7" (178 mm) serrated tip, broad grip forceps | Electron Microscopy Sciences | 77937-28 | Example of blunt forceps needed for dissection |
Straight and curved dissection scissors | Electron Microscopy Sciences | 72966-02 and 72966-03 | Examples of scissors needed for dissection |
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