This work was supported by the National Institutes of Health (Grants R00-HD068130 to S.W.) and the Baltimore Diabetes Research Center: Pilots and Feasibility Grant (to S.W.).

Here, we present detailed protocols for DHT pellet preparation and insertion, and for reproductive and metabolic testing. The mice used in this study were a mixed background (C57/B6, CD1, 129Sv) and were maintained with food and water ad libitum in a 14/10 h light/dark cycle at 24 &#176;C in the Broadway Research Building animal facility at the Johns Hopkins University School of Medicine. All procedures were approved by the Johns Hopkins University Animal Care and Use Committee.
1. Create PCOS-like Mouse Model
<ol>
	<li>DHT pellet preparation
	<ol>
		<li>Autoclave silastic tubing to sterilize. Cut silastic tubes to a length of 15 mm with a razor blade;</li>
		<li>Seal one side by injecting medical adhesive silicone into the tube with a 20 G&#160;blunted needle attached to a 3 mL syringe. Plunger should be withdrawn from the syringe and then adhesive inserted into the reservoir. The plunger is reinserted and adhesive pushed down until it begins to emerge from the needle. The blunted needle is made by cutting the sharp tip side of 20 G needle with any strong scissors. The length of silicon in the tube should be more than 2 mm to allow for post-production trimming;</li>
		<li>Dry overnight; check for air bubbles on the sealed side. Those with no air bubbles are used for DHT pellets. Others can be used for no-DHT control pellets.</li>
		<li>Wear gloves, mask, goggles and lab coat before making DHT-pellet in a hood to avoid DHT exposure to skin.</li>
		<li>Pour DHT powder into a plastic weigh boat and press the open side of the adhesive capped tubes (produced in A1.1) into the DHT powder.</li>
		<li>DHT powder can be tamped down with a large paper clip that is straightened.&#160; Continue until DHT reaches a height of 4 mm (or desired length). Check the length of DHT with a ruler, and seal the open side with silicon, dry overnight.</li>
		<li>Cut each sealed side to make the length of silicon 2 mm long. The total length of pellet will be 8 mm.</li>
		<li>Seal both side of empty silastic tubing as control (no-DHT) pellet.</li>
		<li>Keep pellets in a 50 mL conical tube wrapped with foil (to prevent light exposure) at room temperature until &#160;use. The DHT pellets maintain full efficacy for at least 3 months of storage.</li>
	</ol>
	</li>
	<li>DHT insertion and replacement
	<ol>
		<li>Up to 20 DHT pellets or control pellets are submerged separately in a 50 mL conical tube with 30 mL sterile 0.9% saline for 24 h at 37 &#176;C for equilibration just before insertion.</li>
		<li>Prior to surgery, the surfaces and gloves are disinfected with clidox and the work surfaces are covered &#160;&#160;with clean plastic backed absorbent paper (surgical pad).&#160; All instruments that come in contact with animals are decontaminated prior to entering the animal facility by autoclaving.&#160; Instruments will be allowed to dry and cool prior to use.</li>
		<li>2-month-old female mice are used (4&#8211;5 mice/cage). Mice&#160;are injected intraperitoneally with Xylazine (3.5 mg/kg bw)&#160;and Ketamine (78.8 mg/kg bw) using an insulin syringe. Calculations for mixing and dosing anesthesia is in Table 1.&#160;</li>
		<li>After adequate anesthesia is achieved, as measured by loss of toe reflex and slowed breathing, the mouse will be prepped for surgery.</li>
		<li>Disinfect the skin of the area with betadine using sterile gauze and clean with 70% ethanol. &#160;The area will again be painted with betadine.</li>
		<li>Cut a hole around 5 mm length with scissors under the skin near the neck. Using a 10 G trochar, make a small tunnel (15 mm) in the rostral direction. The pellet is inserted dorsally with the trochar.</li>
		<li>The opening is then sealed with surgical adhesive. Manually approximate the wound edges with forceps and gentle brushing strokes to apply a thin film of liquid adhesive to the approximated wound edges. Buildup 3 thin layers of adhesive to ensure the adhesive is evenly distributed over the wound. The adhesive should extend 1 cm on each side of the apposed wound edges. Suture or surgical clips can also be used to close the hole. Put mice back into cage, individually housed, with a heat pad for recovery. Replace pellet every 4 weeks to maintain a constant level of androgen exposure. The original pellet will be removed and new pellet will be inserted as described in A1.15 in the similar position.</li>
		<li>Test estrous cyclicity after 3 days of DHT insertion. Estrous cycle stage is assessed by vaginal cytology.&#160; Vaginal cells are collected for 16 consecutive days by using a p10 pipettor to squirt sterile saline (around 10 &#181;L) into the vaginal cavity and then withdrawing the saline with the same sterile pipette tip.&#160; Vaginal cells sloughed from vaginal wall mix with saline and are collected.</li>
		<li>Spread saline with cells onto a labeled slide. Each slide can contain six samples. Slides are labeled to note which sample is placed in each of the six locations.</li>
		<li>After the saline has completely dried out on the slide, put slides into a container with 100% ethanol to fix cells. Slides should be fixed for at least 5 min, but can remain in fixative indefinitely until further use.</li>
		<li>Put slides into staining solutions for 1 min each for buffer B&#160;and C. Wash slides with tap water and dry at room temperature.</li>
		<li>Examine cell morphology under a 10X objective light microscope.&#160; Cell morphology to distinguish proestrus (P), estrus (E), metestrus (M) and diestrus (D) is described in references27,28,29.</li>
		<li>Count the days in each stage and divided by the total number of days to calculate percent time at each stage.</li>
		<li>Test glucose tolerance at 21 days after DHT insertion by fasting the mice overnight (16 h) and injecting 2 g/kg body weight of 20% dextrose intraperitoneally. For example, if a mouse body weight is 25 g, this mouse will be injected with 250&#160;&#181;L&#160;of 20% dextrose with a 50 cc (0.5 mL) insulin syringe. Glucose levels are assessed by glucometer and test strips by sampling blood from tail vein at 0, 15, 30, 60, 90, 120 min. This is described in detail elsewhere30.</li>
	</ol>
	</li>
	<li>Blood collection
	<ol>
		<li>Collect blood at various days after DHT insertion (we limit collection volume to 100 &#181;L for steroid and 30ul for LH/FSH assay) between 9 and 10 am by submandibular vein bleeding with lancet. This is described in11.</li>
		<li>Centrifuge blood at 6,000 x&#160;g at 4 &#730;C for 10 min, and collect the serum layer into another 1.5 mL tube that can be stored at -80 &#176;C until assay.</li>
	</ol>
	</li>
</ol>
2. Assess Reproductive Profiles of Female Offspring from Chronically DHT Inserted Dams
<ol>
	<li>Mating
	<ol>
		<li>The female mouse to be tested is moved into the cage with a proven fertile male (that has&#160; previously had pups with a female mouse) at 15 days following pellet insertion.&#160;</li>
		<li>Document dam&#8217;s body weight every week to determine if the mouse is pregnant. Dam weight increases of more than 3 g in a week indicates pregnancy.</li>
		<li>Collect blood of dams the second week after we observe increased body weight of dams greater than 3 g from the week before.&#160;</li>
	</ol>
	</li>
	<li>Puberty assessed by vaginal opening and first estrus
	<ol>
		<li>Check vaginal opening by visual inspection every day after pups are weaned at 21 post-natal day (PND), measure the anogenital distance and record the age of vaginal opening.</li>
		<li>Once vagina opens, collect vaginal cells daily, as described in 1.2.8&#8211;1.2.11&#160;to check estrous cyclicity.</li>
		<li>Observe cell morphology as described 1.2.12, and the first estrus is defined as the date that all cells are cornified epithelial cells.&#160;</li>
	</ol>
	</li>
	<li>Body weight and ear punch
	<ol>
		<li>Dip the lancet tip into the tattoo paste and mark each pup with tattoo on the toe as&#160; &#160;described in reference12 at 7 PND, and weigh mice every 7 days until 70days of age.</li>
		<li>Ear punch mice to number them using system in Figure 1 between 12 to 16 PND. To distinguish sex, examine ventral side of body, females will have visible nipples.</li>
		<li>Collect blood at 21, 26, 70 PND after birth as described above in 1.3.1</li>
	</ol>
	</li>
	<li>Hormonal Assay
	<ol>
		<li>DHT levels in blood serum are measured by both enzyme-linked immunosorbent assay (ELISA) and by liquid chromatography tandem mass spectrometry (LC-MS)23,24,31,32. T is measured by LC-MS, or a rat/mouse ELISA that has been validated by the University of Virginia Ligand Assay Core in the Center for Research in Reproduction23,24.</li>
	</ol>
	</li>
</ol>

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Nothing to disclose.

Hyperandrogenism is a key feature of PCOS. The serum DHT levels (two fold higher in DHT mice than in no-DHT mice) used in this protocol are lower than those reported by other investigators in previous studies and are calibrated to proportionally mimic women with PCOS5,19,20,21. Unlike other models, this 2-fold DHT model does not alter the body weight and whole body composition compared with no-DHT mice for up to 3.5 months after DHT insertion23,24. These adult DHT implanted mice are maintained by continual exogenous DHT. Although whole body composition is not altered, alteration of the structure and function of the adipocytes has been observed in lean PCOS women20. A careful examination of different depots is warranted. We observed impaired estrous cyclicity within one week after DHT insertion15 and reduced fertility during a 3 month fertility evaluation23.
The small subset of DHT treated female mice that were able to get pregnant and have offspring provided an opportunity to assess the impact of pre-gestational, gestational and nursing hyperandrogenemia on fetal development. In order to get a properly controlled experiment, multiple breeding females are required which necessitates that at least 10 mating pairs be established. DHT-daughters are only exposed to DHT during gestation and before 21 PNA. DHT-daughters showed reduced body weight compared to no-DHT daughters, indicating that obesity or overweight is not a confounding variable in the observed physiological effects15.&#160;&#160;
In initial studies, we validated the serum DHT levels over time. We found no significant differences in serum DHT at 1, 2, 3, and 4 weeks23,24. DHT levels declined after 4 weeks, therefore, we replace DHT pellet every 4 weeks. We do not observe reduced effects of DHT even they have been stored at room temperature for 3 months. It is important to incubate pellets in saline for 24 h just before insertion. This step is critical for even release of DHT from the pellet (DHT release slowly through the silastic tubing). Estrous cyclicity can be examined after 3 days of DHT insertion, and vaginal smear can be also examined directly in the saline without dry and stain under a light microscope after you familiar with the cell types27.
Metabolic phenotype can be assessed at, or after, 2 weeks (14 days) of DHT insertion. However, the effects of DHT on metabolism at the end of the fourth week (28 days) are slightly attenuated, though no attenuation is observed for reproductive dysfunction. Therefore, we assess metabolism function normally at the end of 2, 3, 5 , 6, 7 weeks (14, 21, 35, 42, and 49 days, 2 times total insertion). The production of slow release steroid pellets in the lab is a mature technique that has been widely adopted by different laboratories33,34.&#160; It represents a cost-effective alternative to commercially available products, which can exceed $50/pellet (90 day DHT pellet, 5mg/pellet, IRA, NA-161). The commercial product does have the advantage of not needing to be replaced for up to 90 days, while the pellet described in this report needs to be replaced every 28 days. For large-scale studies, investigators may find it more economical to produce their own pellets even with the added work required to replace pellets every 4 weeks.&#160;
As observed by others, models with chronic DHT implantation after birth do not show increased LH pulsatile frequency and this may point out different pathological mechanisms of elevated androgen induced reproductive function between developmental and late-onset acquired hyperandrogenism. With this protocol, we are able to investigate female offspring from chronically hyperandrogenic dams. We can leverage this model to try to fully understand the reproductive (e.g. follicle development, ovulation (corpora lutea), and fertility) and metabolic consequences (e.g. glucose or lipid metabolism, body composition, adipose depot distribution). Our protocol adds new tool to investigate elevated androgen induced reproductive and metabolic dysfunction; and dissociates the pathophysiology caused by elevated androgen from those that could be due to obesity. This model can be used to explore the tissue specific effects of elevated androgens in females. In addition, the methodology reported here for DHT pellet production can be easily applied to the study of other steroid hormones.&#160;&#160;

Hyperandrogenism is the hallmark of polycystic ovary syndrome (PCOS) according to NIH criteria and of&#160;the Androgen Excess and PCOS (AE-PCOS) Society. Women with PCOS have difficulty getting pregnant and have increased risk of pregnancy complications1. Even if they get pregnant, their female offspring have an adverse health outcomes2,3. Animal models have been developed using various strategies4,5,6,7,8,9,10,11,12 and exhibiting many features of PCOS (anovulation, and or impaired glucose and insulin tolerance) with increased body weight and obesity associated with enlarged adipocyte size and increased adipocyte weight. There are two major strategies to produce animal models that are used to study PCOS. One is treatment with high levels of androgens directly (exogenous androgen injection/insertion) or indirectly (such as blocking androgen conversion to estrogen with aromatase inhibitor) after birth13. Another is by fetal hyperexposure of androgens during gestation14,15 to study the offspring. For example, female offspring from rhesus monkey16,17, sheep18, and rodents exposed to male levels of androgen during the intrauterine period develop PCOS-like traits later in life. These models significantly enhanced our understanding of elevated androgen effects, and fetal programing, and uterine environmental effects. However, these models have their own limitations: 1) animals develop obesity and it is therefore difficult to separate the effects of hyperandrogenemia from obesity induced reproductive and metabolic dysfunction; 2) before pregnancy, women with PCOS already exhibit high levels of androgen, thus oocytes have been exposed to androgen excess before fertilization; 3) the pharmacological doses of testosterone (T) or dihydrotestosterone (DHT) used after birth or during gestation may not reflect the androgen environment of PCOS. Testosterone and DHT levels have been measured in ovarian follicular fluid and/or serum, and testosterone and DHT levels are 1.5 to 3.9 fold higher in women with PCOS5,19,20,21,22,23 compared to unaffected women. We created an adult mouse model23,24,25 that develops reproductive and metabolic dysfunction within two weeks of the initiation of chronic DHT exposure from insertion of a pellet with 4mm length of crystal DHT powder (total length of pellet is 8mm). This model produces serum DHT levels that are about 2-fold higher (referred to as 2xDHT) than that of control mice without DHT treatment. The 2xDHT mice do not&#160;exhibit alterations of basal serum estradiol, testosterone,&#160;LH and do not&#160;develop obesity, and&#160;show similar ovarian weight, serum levels of cholesterol, free fatty acids, leptin, TNF&#945; and IL-623,24,25 relative to controls even up to 3.5 months after DHT insertion23,24,25. Additionally, by mating females that have already developed features of PCOS, we can study the impact of a hyperandrogenic maternal environment on the metabolic and reproductive health of the offspring15.
This new paradigm (relevant to NIH and AE-PCOS Society criteria) models the disease by producing relatively similar levels of androgens to those of women with PCOS 2- to 3-fold higher testosterone or DHT levels compared to unaffected women. However, this model is maintained by continual exogenous DHT and not from programmed endogenous hyperandrogenism once DHT is withdrawn. The overall goal of this article is to focus on 1) how to make the DHT pellet; 2) how to generate a lean-PCOS like mouse model; 3) strategies to evaluate female offspring from these dams. Other measurements and assessment of phenotypes are not addressed in this manuscript but can be found in5,15,23,24,25,26.

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a hyperandrogenic mouse model to study polycystic ovary syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Serum DHT levels and Glucose tolerance test
DHT levels were measured from collected serum by both ELISA and by LC-MS according to protocol 1.24&#8211;1.25, and 2.9, 3.0. The DHT absolute values are different between mass spectrometry and ELISA, however, the relative fold (around 2-fold) of DHT vs no-DHT insertion is similar from both assays and across experiments15,23,24 (Figure 2A).&#160; DHT levels are significantly increased from preconception through pregnancy in both DHT and no-DHT mice, however, DHT levels are 2-fold higher in the DHT mice compared to the no-DHT mice at both pregestational (one day before mating) and gestational (around 14 days) time points (Figure 2B). Since absolute levels of DHT differ between ELISA and LC-MS, we calculate relative levels (fold change: DHT level with DHT insertion vs no-DHT insertion) within assays. Female mice with DHT showed impaired glucose tolerance relative to no-DHT (Figure 3) at 2 weeks following DHT insertion according to protocol 1.23.
Female offspring body weight and puberty
While generally infertile, some dams get pregnant and have pups (around 30% pregnancy rate, so testing fertility of 10 dams we will usually get 3 pregnant dams, refer to protocol 2.1). We therefore can evaluate chronic androgen effects in dams on the female offspring. Female offspring from DHT inserted dams are called DHT-daughters, and those from no-DHT inserted dams are called no-DHT daughters. We observed no difference between no-DHT and DHT-daughters for the age of vaginal opening. However, the first estrus of DHT-daughters is significantly delayed (day 35 for no-DHT daughter; day 42 for DHT-daughter). This is associated with reduced body weight at both 35 and 42 PND in DHT-daughters relative to no-DHT daughters (Figure 4; refer to protocol 2.4&#8211;2.7).
Hormonal levels and estrous cyclicity in female offspring
Testosterone levels are not altered at 21 PND, but are reduced at 26PND. However, testosterone is increased at 70 PND (Figure 5, refer to protocol 2.9&#8211;3.0). Adult DHT daughters showed disrupted estrous cyclicity compared to adult no-DHT daughters, experiencing significantly longer times in M/D and less time in P and E (Figure 6A, B; according to the method in protocol 1.17&#8211;1.22).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58379/58379fig1.jpg" /> 
Figure 1: Mouse identification. Within one cage, mouse is ear punched at different position to represent the mouse number. Mouse #1 to 3 is punched on the right ear, and #4 to 6 is punched on the left ear viewed&#160; dorsally. If mouse is punched on ears at both #1 and #4 position, it is #7; at 2 and 5, is #8; at 3 and 6 is #9; at the middle of left ear is #10; at 1 and 10 position is #11; etc. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58379/58379fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58379/58379fig2.jpg" /> 
Figure 2: Serum DHT levels. Serum DHT levels. (A) Serum DHT levels are measured by both ELISA and Mass-Spectrometry. Although the absolute values are different, the two assays showed similar fold differences between DHT and no-DHT treated mice. (B) Serum DHT fold change relative to non-DHT levels at preconceptional and gestational time points. No-DHT (open bars) and DHT-implanted (black bars) female mice before (one day before mating) and during pregnancy (around 14 days of gestation). Values are mean &#177;S.E.M. N = 5-8 per group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58379/58379fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58379/58379fig3.jpg" /> 
Figure 3: Glucose tolerance test (GTT). No-DHT and DHT implanted mice were fasted overnight and glucose (2 g/kg BW) was injected intraperitoneally, and tail blood glucose was measured at different time points. DHT treated mice showed significantly increased glucose levels between 30 to 120 min compared to no-DHT treated mice. Values are mean &#177;S.E.M. N = 4-12 per group. * P &#60;0.05 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58379/58379fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58379/58379fig4.jpg" /> 
Figure 4: Pre-pregnancy maternal DHT treatment resulted in reduced body weight in female offspring at 35 and 42 PND. Body weight (Y-axis) was measured on post-natal days as shown (X-axis). DHT-exposed offspring: black bar; no &#8211;DHT offspring: open bar. Values are mean &#177;S.E.M. N = 9-14 per group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58379/58379fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58379/58379fig5.jpg" /> 
Figure 5: Serum DHT levels. Blood was collected in the morning on PND 21, 26, 70 in the morning. Serum DHT levels (Y-axis) from female offspring of no-DHT daughters (open bars) and DHT-daughters (black bars). Values are mean &#177;S.E.M. N = 5-11 per group. This figure has been modified from reference2 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58379/58379fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58379/58379fig6.jpg" /> 
Figure 6: Chronic maternal androgen excess leads to disturbed cyclicity in adult daughters.&#160;(A) Representation of estrous cyclicity of female offspring. (B) The percentage of time spent at each estrous stage (Y-axis) during 15 days (X-axis) measured by cytological examination of vaginal cells. Values are mean &#177;S.E.M. N = 5-9 per group. The estrous cycle stage (Y-axis). M/D: met/diestrus; P: proestrus; E: estrus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58379/58379fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/58379/58379tbl1.jpg" /> 
Table 1: Ketamine/xylazine cocktail.

Watch this Scientific Journal Video about A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome at JoVE.com

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

a-hyperandrogenic-mouse-model-to-study-polycystic-ovary-syndrome

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11.1K Views.  Johns Hopkins University School of Medicine. We describe the development of a lean PCOS-like mouse model with dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Video: A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

A Mouse Distraction Osteogenesis Model

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the following three phases: the latency phase after osteotomy and placement of the external distractor; the distraction phase, wherein the separated bone ends are gradually and continuously distracted; and the consolidation phase. This custom-made distractor used for DO is comprised of two incomplete acrylic resin rings and an expansion screw. The process was initiated by making a mold with silicone impression material and then creating the custom-made distractor. Dental resin was poured into the formwork made of silicone impression material, and it was allowed to polymerize to create the incomplete resin rings required for the custom-made distractor. These rings were fixed with an expansion screw using transparent resin. The custom-made distractor created via this approach was attached to the tibia of mice. The tibia was fixed to the device using one pair of 25 G needles proximally, one pair of 27 G needles distally, and acrylic resin. After a latency period of 5 days, distraction was initiated at a rate of 0.2 mm/12 h. The lengthening was continued for 8 days, resulting in a total gap of 3.2 mm. The mice were sacrificed 4 weeks after distraction. Bone formation in the distraction gap was confirmed using both radiography and histology.

We present a mouse tibial distraction osteogenesis model developed using a custom-made distractor. The use of a mouse as an analysis target is advantageous for advancing research.

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...