oral delivery of transformed yeast cells into the mouse intestinal immune system

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

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 2,500 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 1-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 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 gavaged 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 serosa 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&#39;s patches by looking for 1 to 2-millimeter roughly circular patches of opaque tissue along the small intestine. Then, using curved dissection scissors, cut away the dome of the Peyer&#39;s patch, leaving margins to ensure that none of the surrounding tissue is collected.
Transfer the dissected Peyer&#39;s patches into complete IMDM. Then, pour the solution with suspended Peyer&#39;s patches onto a 40-micrometer cell strainer. After washing the Peyer&#39;s patches with complete IMDM, use a plunger from a 1-milliliter syringe to gently break up the tissue and collect the cells in a 50-milliliter tube. Pellet the strained cells at 1,800 RPM for seven minutes. Then wait and analyze the cells according to the text protocol.

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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SmartSpec 3000 Spectrophotometer</td>
			<td>BioRad</td>
			<td>170-2501</td>
			<td>Example of spectrophotometer for determining cell concentration and OD600 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</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 rowspan="2">Example of replica plating stand and pads</td>
		</tr>
		<tr>
			<td>Velveteen squares</td>
			<td>Fisherbrand</td>
			<td>09-718-2</td>
		</tr>
		<tr>
			<td>L shaped sterile cell spreaders</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 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 g, 20 gauge or larger straight or curved gavage needles may be used</td>
		</tr>
		<tr>
			<td>1mL 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&#34; (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</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>
	</tbody>
</table>

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.

<img alt="Figure 1" src="/files/ftp_upload/22271/22271_Figure1.jpg" /> 
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.
<img alt="Figure 2" src="/files/ftp_upload/22271/22271_Figure2.jpg" /> 
Figure 2. Survival c...

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

Begin with an oral tube containing a suspension of genetically transformed, auxotrophic yeast that are unable to produce uracil nucleobase.
Insert the oral tube into the mouth of a restrained mouse and guide it down to the stomach.
Administer the yeast suspension directly into the stomach, allowing it to eventually reach the small intestine.
The small intestine contains epithelial cells, with underlying Peyer&#39;s patches rich in immune cells.
Microfold cells, specialized intestinal epithelial cells near the Peyer&#39;s patches, engulf the yeast cells and internalize them into vesicles.
These vesicles move across the cells and release yeast cells into the underlying Peyer&#39;s patch.
Isolate the Peyer&#39;s patch from the sacrificed mouse intestine and obtain a uniform cell suspension.
Culture this cell suspension on a yeast-selective medium containing uracil.
Auxotrophic yeast utilizes the uracil and multiplies to form colonies, confirming the delivery of transformed yeast cells into the mouse intestinal immune system.

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视频: 将转化的酵母细胞口服递送到小鼠肠道免疫系统中 - 实验

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.

新生儿小鼠益生菌的研究

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

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免疫与感染

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

前体内分析研究胃 肠道的 念珠菌血脂磷

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

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

成績單

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