Funding: This work was supported by a Stuart H.Q. &amp; Victoria Quan Predoctoral Fellowship (RMW), NIH Pioneer Award (RAS)

General Health Exam
A more comprehensive treatment of general health exams can be found in Jacqueline Crawley&rsquo;s excellent behavioral phenotyping resource, What&rsquo;s Wrong with my Mouse? 1. We first use a modified observational test battery to assess the general health of the subject. The battery includes items from Samuel Irwin&rsquo;s comprehensive assessment protocol2 to quickly identify those subjects that will be unsuitable for further testing. In brief, the modified battery identifies animals that are physically abnormal (eg. severely under- or over-weight, poorly groomed, dyspneic) or that exhibit abnormal posture, locomotion or behaviors. These all could be manifestations of illness. Any initial evaluation that similarly identifies unhealthy subjects is appropriate.
It is also important to note that animals are used humanely both during the general health exam as well as during subsequent tests. No anesthesia or analgesia is used as these tests do not incorporate any potentially painful procedures.
Olfactory Sensitivity Testing
This test is designed to detect a difference in olfactory sensitivity between two groups of mice (eg. a wild type and a mutant group of animals). In this test, multiple dilutions of a scent are made. A na&iuml;ve animal then investigates a single dilution of this scent and the total exploratory time (within 3 minutes) is recorded. Multiple dilutions of the scent are tested on different, na&iuml;ve subjects and the total exploratory time is used to generate dose response curves for each group of mice. Two researchers should be involved in testing, with one blind to subject genotypes and/or condition.
Primary researcher
<ol>
<li>Obtain four clean cages (8 H x 10 W x 18 L, in inches), all without bedding. Line up the four cages in the biosafety cabinet.</li>
<li>Set up partitions between each arena using opaque filter paper. Place a black poster board across the final partition such that the black side lines the side and bottom of the cage. Cover the top of the arena with a clear piece of plexiglass to allow light to illuminate the arena. (a) <img src="/files/ftp_upload/old/949_2009_1_14_8/figure1new.png" alt="Figure 1" />(b)<img src="/files/ftp_upload/old/949_2009_1_14_9/image15.png" alt="Figure 1.5" /> Figure 1. Arena Set-Up. (a) Properly draped, empty arena (b) Arena with subject and scented filter paper. </li>
 
<li>Begin habituating the subjects. Place the first subject in the first cage for habituation. After 15 minutes, move the first subject to the second cage. Clean the first cage with Quatricide TB. Next, place the second subject in the first, cleaned cage. Continue this process with subsequent subjects. Ultimately, each subject should be habituated in all three cages before being placed in the arena (fourth and final cage) for another 15 minutes of habituation prior to testing.</li>
</ol>
Blind researcher
<ol>
<li>Make an appropriate number of 2 inch by 2 inch squares of filter paper for scent samples.</li>
<li>Using the pre-selected scent, appropriately dilute it. For hydrophobic samples such as peanut butter, use oil as solvent; for all others, use distilled water. Vortex.</li>
<li>Set a digital video recording device at the far end of the biosafety cabinet such that the entire cage end is in focus. Take a preliminary video to ensure parameters are adequate.</li>
<li>Record the order in which dilutions will be tested, taking care to randomize the order of the trials.</li>
<li>Record subject's litter and identification numbers.</li>
<li>Once the subject has been habituated for 15 minutes in the arena, begin preparation for the first trial. Pipette the first dilution up and down in the conical tube before pipetting it onto a filter paper square. The filter paper surface should optimally be ~85% covered (approximately 0.50 mls of solution). Use a kimwipe to make sure the filter paper is not overly saturated.</li>
<li>Begin video recording.</li>
<li>Place the filter paper square in the cage on the opposite end of the animal's current position.</li>
<li>Begin timing.</li>
<li>After 3 minutes, stop the recording and remove the diluted scent.</li>
<li>If only one scent (eg. cinnamon) is to be tested, remove the first test subject from the arena. Repeat steps 6-11 for each animal, appropriately diluting the scent for each subject. If more than one scent is to be tested, however, do not remove the test subject from the arena. Instead, begin timing.</li>
<li>Appropriately dilute the next scent using the same methods as listed above.</li>
<li>After 1 minute, begin video recording. Place the filter paper square with the dilution of the next scent in the cage on the opposite end of the animal's current position.</li>
<li>Repeat steps 6-13 for each scent, noting the video file number for each trial.</li>
<li>Repeat steps 6-14 for each subject. <img src="/files/ftp_upload/old/949_2009_1_14_10/image2new.png" alt="Figure 2" /> Figure 2. Representative results for a session of olfactory sensitivity testing. Each mouse from either the control group (Group A) or the experimental group (Group B) was exposed to one of five different dilutions of cinnamon extract on filter paper for one, three minute session. Total time spent exploring the filter paper was assessed. Mice in Group A have a greater olfactory sensitivity than those in Group B.</li>
</ol>
Olfactory Preference Testing
This test is designed to identify specific detection deficiencies, namely the ability to sense attractive or aversive scents. The following procedure borrows liberally from the protocol of Kobayakawa, K., et al.3. Again, two researchers should be involved in testing, with one blind to subject genotypes and/or condition. The total exploratory time is again recorded (upon video viewing) for various scents. Those scents with total exploratory times greater than water are designated as &ldquo;attractive&rdquo; while those with times less than water are termed &ldquo;aversive&rdquo;. The researcher then considers whether the same scents are &ldquo;attractive&rdquo; or &ldquo;aversive&rdquo; for different test subjects.
Please follow steps 1-3 in the Olfactory Sensitivity Testing Section for the primary researcher.
Blind Researcher
The procedure for the olfactory preference testing is the same, with slight modifications of steps 2 and 4. Please follow steps 1-15 in the Olfactory Sensitivity Testing Section for the blind researcher, with the following changes: For step 2, using the pre-selected scents (a combination of both attractive and aversive scents), make appropriately diluted solutions of each in conical tubes. For hydrophobic samples such as peanut butter, use oil as solvent; for all others, use distilled water. Vortex. For step 4, rather than recording the dilution to be tested, record the order of the varied scents that will be tested, randomizing the order of the trials.
This test is designed to identify specific detection deficiencies, namely the ability to sense attractive or aversive scents. The following procedure borrows liberally from the protocol of Kobayakawa, K. et al.3. Again, two researchers should be involved in testing, with one blind to subject genotypes and/or condition. The total exploratory time is again recorded (upon video viewing) for various scents. Those scents with total exploratory times greater than water are designated as "attractive", while those with times less than water are termed "aversive". The researcher then considers whether the same scents are "attractive" or "aversive" for different test subjects.
Please follow steps 1-3 in the Olfactory Sensitivity Testing Section for the primary researcher.
Blind Researcher
The procedure for the olfactory preference testing is the same, with slight modifications of steps 2, 4 and 13. Please follow steps 1-15 in the Olfactory Sensitivity Testing Section for the blind researcher, with the following changes: For step 2, using the pre-selected scents (a combination of both attractive and aversive scents), make appropriately diluted solutions of each in conical tubes. For hydrophobic samples such as peanut butter, use oil as solvent; for all others, use distilled water. Vortex. For step 4, rather than recording the dilution to be tested, record the order of the varied scents that will be tested, randomizing the order in each trial. For step 13, place the filter paper square with the next scent in the cage on the opposite end of the animal's current position.
<img src="/files/ftp_upload/old/949_2009_1_14_11/image3new.png" alt="Figure 3" />
Figure 3. Representative results for a session of olfactory preference testing. Each mouse was exposed to each of the different scents on filter paper for one, three minute session. Total time spent exploring the filter paper was assessed. Peanut butter and vanilla are attractive scents (exploratory timescent &gt; exploratory timewater) and 2-Methylbutyric Acid is aversive (exploratory timescent &lt; exploratory timewater).
Scoring of the Olfactory Tests
After acquiring all of the olfactory sensitivity and preference videos, it is necessary to analyze the total time spent exploring each dilution/scent. Have the blind researcher analyze all of the videos for consistency.
<ol>
<li>Preview the video, noting the following: a) when the filter paper is placed in the arena, b) when the mouse approaches the filter paper, c) when the mouse investigates the filter paper and d) when three minutes have elapsed (from when the filter paper was placed in the arena). Knowing when to focus on the behavior of the mouse makes it easier to accurately score the total time spent exploring upon review.</li>
<li>Review the video. Note the time when the mouse first investigates the filter paper (latency).</li>
<li>Using a stopwatch, time all investigations within three minutes of placing the filter paper in the arena. The time of investigation is defined as any time when the mouse&rsquo;s nares are &lt; 1 mm from the filter paper. It is important not to include any of the time when the mouse is in close proximity to the filter paper, but is not actively investigating it. Do not include bouts of chewing and licking the paper or bouts of sitting on the paper in the total exploratory time. It is necessary to adhere closely and consistently to these rules to accurately analyze olfactory behavior.</li>
<li>Record latency time, the total time spent investigating the filter paper and video file number.</li>
</ol>

<ol>
	<li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What&amp;rsquo;s+Wrong+with+my+Mouse?+Behavioral+Phenotyping+of+Transgenic+and+Knockout+Mice.">What&amp;rsquo;s Wrong with my Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice.</a> , 42-60 (2007).</li><li>Irwin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+observational+assessment:+Ia.+A+systematic,+quantitative+procedure+for+assessing+the+behavioral+and+physiologic+state+of+the+mouse.">Comprehensive observational assessment: Ia. A systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse.</a> Psychopharmacologia. 13, 222-257 (1968).</li><li>Kobayakawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+versus+learned+odour+processing+in+the+mouse+olfactory+bulb.">Innate versus learned odour processing in the mouse olfactory bulb.</a> Nature. 450, 503-508 (2007).</li></ol>

The aforementioned tests allow for the quick and reliable assessment of basic olfactory function. It is not to be overlooked, however, that this reliability requires the proper selection and preparation of subjects for testing. Animals that are ill or otherwise incapacitated are often inconsistent test subjects. Also, habituation of the test subjects is key. We have found that the use of multiple cages during the habituation process allows the animals to become comfortable with experimenter handling and changing surr...

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<td>2-Methylbutyric acid</td>
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<td>Pure Vanilla Extract</td>
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<td>Peanut Butter (Skippy, Extra Crunchy)</td>
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olfactory behavioral testing in the adult mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Watch this Scientific Journal Video about Olfactory Behavioral Testing in the Adult Mouse at JoVE.com

Olfactory Behavioral Testing in the Adult Mouse

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

olfactory-behavioral-testing-in-the-adult-mouse

Research

JoVE Journal

Biology

19.4K Views.  Dana Farber Cancer Institute. Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

Video: Olfactory Behavioral Testing in the Adult Mouse

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...