We thank Dr. Vicki Rosen for financial support and guidance with the project. We would also like to thank the veterinary and IACUC staff at the Harvard School of Medicine for consultation regarding sterile technique, animal well-being, and the materials used to develop this protocol.

All animal experiments described were approved by the Institutional Animal Care and Use Committee of the Harvard Medical Area. 12-week-old C57BL/6J mice (males and females) were used in this protocol. C57BL/6J male and female mice achieve peak bone mass around 12 weeks of age with femurs wide enough to fit a stabilizing pin, making them an appropriate strain to use for this protocol15.
1. Preparation for the surgery
<ol>
	<li>Autoclave the surgical equipment, including surgical scissors, straight forceps, curved forceps, surgical clamps, and diamond cutting wheel (see Table of Materials) to minimize the risk of infection.</li>
	<li>Place a clean mouse cage on a heating pad to facilitate post-surgical recovery. Set the heat pad to reach a temperature between 37-45 &#176;C.</li>
	<li>Place the mouse under anesthesia using an isoflurane chamber. Set the induction oxygen flow at 2 L/min, the isoflurane induction at 2-4%, the maintenance nose cone oxygen at 2 L/min, and the maintenance isoflurane is 1.4%.</li>
	<li>Confirm that the mouse&#39;s breathing is stable and they do not respond to a toe pinch. Apply a thin layer of ophthalmic ointment on each eye to prevent corneal scratching. Transfer the mouse to a sterile pad and maintain anesthesia using a nose cone to deliver isoflurane continuously at the same rate as in step 1.3. 
	NOTE: Using 12-week-old mice or older is recommended as femurs from younger mice might be too thin to accommodate a stabilizing pin.</li>
	<li>Inject the mouse subcutaneously with 0.05 mg/kg body weight of slow-release buprenorphine (see Table of Materials).</li>
	<li>Using an electric trimmer, shave a 2 x 2 cm square on both thighs corresponding to the location of the femur.</li>
	<li>Disinfect the shaved area using sterile gauze or swabs to spread a layer of iodine followed by a rinse with 70% ethanol (Figure 1A).</li>
</ol>
2. Surgery
<ol>
	<li>Using a sterile scalpel, make a 5 mm incision in the shaved, disinfected region and peel away the skin to expose the underlying fascia.</li>
	<li>Use straight forceps and fine scissors to delicately grab and cut the fascia directly covering the femur to expose the muscle. A 5 mm cut of the fascia is sufficient to access the underlying muscle.</li>
	<li>Using one pair of straight forceps, gently separate the muscle from the femur with minimal tissue damage.</li>
	<li>Once the femur is visible, slide the curved forceps underneath the femur between the separated muscle and bone. Let the forceps slowly open to maintain muscle separation and secure the femur to facilitate a clean cut. 
	NOTE: The femur must stay exposed and separated from the muscle and skin when the forceps are not held, as shown in Figure 1B.</li>
	<li>Make a transverse cut in the middle of the femur shaft using a handheld saw on the low power setting (See Table of Materials for the blade and Rotary Tool Kit used). 
	NOTE: Once the femur is completely cut, two fracture ends are created: the proximal section (attached to the hip bone) and the distal section (attached to the knee, also known as the stifle joint). Avoid cutting the femur in one single motion. Instead, make 3-5 passes until the femur is completely cut through. This is critical to avoid overheating the surrounding tissue and generating significant bone debris, negatively impacting healing.</li>
	<li>Insert a guide needle (23 G x 1 TW IM, 0.6 mm x 25 mm) (see Table of Materials) in the marrow cavity of the distal section. Use fingers to gently twist the needle as you push to thread through the knee joint (Figure 1C).
	<ol>
		<li>Remove the guide needle from the distal end and repeat on the proximal end, using the same gentle twisting motion to push the guide needle through the hip joint. Leave the guide needle in the proximal end, with its tip emerged from the skin (Figure 1D). 
		NOTE: To stabilize the fracture ends, a guide needle was first used to create a pathway through the fracture ends, and then a stabilizing pin was threaded through this pathway to secure the fracture ends14.</li>
	</ol>
	</li>
	<li>Insert the stabilizing pin (needle, 27 G x 1 &#188;, 0.4 mm x 30 mm) (see Table of Materials) into the tip of the guide needle (Figure 1E). Gently push so that the stabilizing pin enters as the guide needle exits the marrow cavity of the proximal end.
	<ol>
		<li>Discard the guide needle. Use tweezers to hold and align the distal end with the proximal end and continue to thread the stabilizing pin through the distal marrow cavity until it exits the knee joint using the pathway made in 2.6 (Figure 1F). 
		NOTE: The stabilizing pin should now be protruding from the hip and the knee joints.</li>
	</ol>
	</li>
	<li>Using the surgical clamp, pull the tip of the pin to bring the proximal and distal sections close together, so they are barely touching. Realign the fracture ends with forceps if needed, and fold the ends of the stabilizing pin toward the fracture site using the surgical clamp (see Table of Materials).
	<ol>
		<li>Remove the plastic from the base of the needle using wire cutters. Using the clamp, twist both ends of the pin until they are blunt to avoid internal tissue damage. 
		NOTE: The fracture ends are now locked in place so that the mouse can put weight on the injured leg. The pin is secured if the fracture ends are unable to separate. A wire cutter can also be used to blunt the ends. The stabilizing pin must stay in place for the whole duration of the study until the mouse is euthanized. Attempts at dislodging the pin are likely to destabilize the fracture response and cause harm to the animal. The pin can be removed at dissection.</li>
	</ol>
	</li>
	<li>Use straight forceps to reposition the muscle onto the femur. Using curved forceps, pinch the ends of the skin together and close the opening using wound clips. 
	NOTE: Do not close the skin too tightly with the clips or the mouse will avoid putting weight on this leg. Limiting physical loading during recovery could delay the healing process.</li>
	<li>Repeat steps 2.2 to 2.4 on the other leg and close the wound without performing the femur fracture. 
	NOTE: This sham-operated femur serves as the contralateral control.</li>
	<li>Withdraw the isoflurane exposure, place the mouse in the heated cage, and ensure they regain consciousness within 10-15 min.</li>
	<li>Monitor the mouse&#39;s activity and incision site daily for 5 days after surgery for signs of distress or infection. 
	NOTE: If the animal demonstrates signs of pain, is not eating, and/or hesitates to walk on their hindlimbs, consult with veterinary personnel and administer additional analgesic.</li>
	<li>Remove the wound clips 10 days after the surgery. 
	&#8203;NOTE: If the wound does not appear to have closed after 10 days, consult the veterinary personel and IACUC before removing the clips.</li>
</ol>
3. Tissue harvest
<ol>
	<li>Euthanize the mice via CO2 inhalation followed by cervical dislocation. 
	NOTE: This procedure is consistent with the Panel on Euthanasia of the American Veterinary Medical Association.</li>
	<li>Using scissors and forceps, remove the skin from both mouse legs, dislocate the femoral head from the hip bone, and cut adjacent muscle to free the leg. 
	NOTE: Avoid removing too much muscle around the femur as the callus could be dislodged or damaged.</li>
	<li>Avoiding the stabilizing pin, cut through the knee joint to separate the femur from the tibia.</li>
	<li>Dissect around the distal and proximal parts of the femur to expose the ends of the stabilizing pin.</li>
	<li>With a hard wire cutter, cut the folded blunt ends of the pin so only the straight portion of the pin remains. Use forceps to slowly and gently slide the stabilizing pin out of the femur. 
	&#8203;NOTE: If the pin does not easily slide out, do not apply force as this could dislodge the callus and damage the sample. Instead, try to rotate the pin and remove it delicately. Removal of the pin may also be easier after fixation.</li>
</ol>
4. Histology - Alcian Blue / Eosin /Orange G staining
NOTE: Alcian Blue/Orange G/Eosin staining is routinely used to visualize cartilage (blue) and the bone (pink). The cartilage area can be quantified as a proportion of the total callus area (Figure 2A,B).
<ol>
	<li>Fix femurs in 10% neutral buffered formalin overnight at 4 &#176;C.</li>
	<li>Wash the fixed samples in phosphate-buffered saline (PBS).</li>
	<li>Place samples in 0.5M EDTA, pH 8.0 on a slowly rotating shaker for 2 weeks. Change EDTA solution every other day to ensure efficient decalcification. 
	NOTE: No specific rpm is required for the shaker; ensure that the liquid flows in the container to cover all the samples. Complete decalcification can be tested by X-ray of the femurs.</li>
	<li>To process the samples for paraffin embedding incubate them in the following solutions (1 h each): 70% EtOH, 95% EtOH, 100% EtOH, 100% EtOH, Xylene, Xylene, Paraffin, Paraffin.</li>
	<li>Embed the samples in paraffin for sectioning.</li>
	<li>Cut 5-7 &#956;m thick longitudinal sections of the fractured femurs using a microtome.</li>
	<li>Deparaffinize the sections by incubating them in 2 baths of Xylene (5 min each).</li>
	<li>Rehydrate the sections by incubating in the following ethanol gradient (2 min each): 100% EtOH, 100% EtOH, 80% EtOH, 70% EtOH.</li>
	<li>Place slides in tap water for 1min.</li>
	<li>Make the solutions of Alcian blue, Acid alcohol, Ammonium water, and Eosin/Orange G for histology as mentioned in Supplementary File 1. 
	NOTE: The suggested volumes of each solution should be sufficient to completely submerge the slides for the staining.</li>
	<li>Place slides in Acid alcohol for 30 s and incubate in Alcian blue for 40 min.</li>
	<li>Wash gently under the running tap until the water is clear for about 2 min.</li>
	<li>Dip the slides rapidly in Acid alcohol for 1 s.</li>
	<li>Rinse as described in step 4.9.</li>
	<li>Incubate in ammonium water for 15 s.</li>
	<li>Rinse as described in step 4.9.</li>
	<li>Place in 95% EtOHfor 1 min and incubate in Eosin/Orange G for 90 s.</li>
	<li>Dehydrate the slides quickly with a single dip in 70% EtOH, 80% EtOH and 100% EtOH.</li>
	<li>Place slides in Xylene for 1 min to clear the slides.</li>
	<li>Apply mounting media and a coverslip for imaging. 
	&#8203;NOTE: For molecular analysis, RNA and protein can be isolated from the callus. Carefully dissect the muscle under a dissecting scope and separate the callus from the underlying bone using a scalpel.</li>
</ol>
5. MicroCT
NOTE: In the later stages of healing, microCT can be performed to image and quantify the mineralization in the hard callus and the fracture gap. In C57BL/6J mice, the callus is usually mineralized and detectable by microCT after 10 days post-fracture (dpf) (Figure 2C).
<ol>
	<li>Fix femurs in 10% neutral buffered formalin overnight at 4 &#176;C. 
	NOTE : MicroCT can be performed on the same femurs used for histology as long as it is done before EDTA decalcification (step 4.3). When using the same femurs for both techniques, perform microCT, retrieve the samples and go to step 4.3.</li>
	<li>Wash the fixed samples in phosphate-buffered saline (PBS) and store in 70% EtOH.</li>
	<li>Perform microCT with an isotropic voxel size of 7 &#956;m at an energy level of 55 kVP and an intensity of 145 &#956;A (see Table of Materials).</li>
	<li>Contour the microCT slices to include the callus and exclude the cortical bone. 
	NOTE: As the callus gets more mineralized with time, the threshold can be adjusted to visualize and measure the callus volume at different stages.</li>
	<li>Obtain the bone volume contained in the callus contours as a measurement of the callus volume. 
	NOTE: Fracture gap can be measured directly on the microCT slices as the distance occupied by the break.</li>
</ol>

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The authors do not have any conflicts of interest to disclose.

The injury model detailed in this protocol encompasses all four significant steps observed during the healing of spontaneous fractures, including (1) pro-inflammatory response with the formation of the hematoma, (2) recruitment of skeletal progenitors from the periosteum to form the soft callus, (3) mineralization of the callus by osteoblasts and (4) remodeling of the bone by osteoclasts.
The surgical procedure described in this manuscript is optimized for adult mice at least 12 weeks old. A 2...

Fractures, breaks in the continuity of the bone surface, occur in all segments of the population. They become severe in people who have fragile bones due to aging or disease, and the health care costs of fragility fractures are expected to exceed $25 billion in 5 years1,2,3,4,5. Understanding the biological mechanisms involved in fracture repair would be a starting point in developing new therapies aimed at enhancing the healing process. Previous research has shown that, upon fracture, four significant steps occur that enable bone to heal: (1) formation of the hematoma; (2) formation of a fibrocartilaginous callus; (3) mineralization of the soft callus to form bone; and (4) remodeling of the healed bone6,7. Many biological processes are activated to heal the fracture successfully. First, an acute pro-inflammatory response is initiated immediately after a fracture6,7. Then, the periosteum becomes activated and expands, and periosteal cells differentiate into chondrocytes to form a cartilage callus that grows to fill the gap left by the disrupted bone segments6,7,8,9. Neural and vascular cells invade the newly formed callus to provide additional cells and signaling molecules needed to facilitate repair6,7,8,9,10. In addition to contributing to callus formation, periosteal cells also differentiate into osteoblasts that lay down woven bone in the bridging callus. Finally, osteoclasts remodel the newly formed bone to return to its original shape and lamellar structure7,8,9,10,11. Many groups developed mouse models of fracture repair. One of the earlier and most often used fracture models in mice is the Einhorn approach, where a weight is dropped on the leg from a specific height12. The lack of control over the angle and the force applied to induce the fracture creates a lot of variability in the location and size of the bone discontinuity. Subsequently, it results in variations in the specific fracture healing response observed. Other popular approaches are surgical intervention to produce a tibial monocortical defect or stress fractures, procedures that induce comparatively milder healing responses10,13. Variability in these models is primarily due to the person conducting the procedure14.
Here, a detailed mouse femur injury model allows for control over the break to provide a reproducible injury and allow for quantitative and qualitative assessment of femur fracture repair. Specifically, a complete breakthrough in the femurs of adult mice is introduced and stabilizes the fracture ends to account for the role physical loading plays in bone healing. The methods for harvesting tissues and imaging the different steps of the healing process using histology and microcomputed tomography (microCT) are also provided in detail.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>23 G x 1 TW IM (0.6 mm x 2 5mm) needle</td>
			<td>BD precision</td>
			<td>305193</td>
			<td>Use as guide needle</td>
		</tr>
		<tr>
			<td>27 G x 1 &#188; (0.4 mm x 30 mm)</td>
			<td>BD precision</td>
			<td>305136</td>
			<td>Use as stabilizing pin</td>
		</tr>
		<tr>
			<td>9 mm wound autoclip applier/remover/clips kit</td>
			<td>Braintree Scientific, INC</td>
			<td>ACS-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue 8 GX</td>
			<td>Electron Microscopy Sciences</td>
			<td>10350</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Millipore Sigma</td>
			<td>AX1303</td>
			<td></td>
		</tr>
		<tr>
			<td>Circular blade X926.7 THIN-FLEX</td>
			<td>Abrasive technologies</td>
			<td>CELBTFSG633</td>
			<td></td>
		</tr>
		<tr>
			<td>DREMEL 7700-1/15, 7.2 V Rotary Tool Kit</td>
			<td>Dremel</td>
			<td>7700 1/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>ThermoScientific</td>
			<td>7111</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine curved dissecting forceps</td>
			<td>VWR</td>
			<td>82027-406</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxulin Gill 2</td>
			<td>Sigma-Aldrich</td>
			<td>GHS216</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Millipore Sigma</td>
			<td>HX0603-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical kit</td>
			<td>VWR</td>
			<td>95042-540</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Sigma-Aldrich</td>
			<td>1625</td>
			<td></td>
		</tr>
		<tr>
			<td>Phloxine B</td>
			<td>Sigma-Aldrich</td>
			<td>P4030</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine Swabs</td>
			<td>PDI</td>
			<td>S23125</td>
			<td></td>
		</tr>
		<tr>
			<td>SCANCO Medical &#181;CT35</td>
			<td>Scanco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slow-release buprenorphine</td>
			<td>Zoopharm</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

transverse fracture of the mouse femur with stabilizing pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

In C57BL/6J mice, a successful surgery completes the healing steps mentioned earlier with little to no local inflammatory response or periosteal involvement in the sham-operated contralateral femur. A hematoma is formed a few hours after surgery, and the periosteum is activated to recruit skeletal progenitors for chondrogenesis. Various cell populations, such as Prx1+ mesenchymal progenitors, can be traced during the repair process using commercially available fluorescent reporter mouse models (<strong class="...

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Transverse Fracture of the Mouse Femur with Stabilizing Pin

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

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Biology

3.3K Views.  Harvard School of Dental Medicine. This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Video: Transverse Fracture of the Mouse Femur with Stabilizing Pin

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

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 ...

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 ...

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

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 ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria&#39;s distribution, fusion, and fission events without staining the cells.

The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAMexcised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive.

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative ...

A Modified Technique for Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of pressure overload in mouse models. The main challenge of this procedure is to clearly visualize the transverse aortic arch and precisely band the target vessel. Classical approaches perform a partial thoracotomy to expose the transverse aortic arch. However, it is an open-chest model that causes a rather large surgical trauma and requires a ventilator during the surgery. To prevent unnecessary trauma and simplify the operating procedure, the aortic arch is approached via the proximal proportion of the sternum, reaching and binding the target vessel using a small self-made retractor that contains a snare. This procedure can be conducted without entering the pleura cavity and does not need a ventilator or microsurgical operation, which leaves the mice with physiological breathing patterns, simplifies the procedure, and significantly reduces operation time. Due to the less invasive approach and less operation time, mice can undergo fewer stress reactions and recover rapidly.

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of ...

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture healing in translational science investigations. Studies of long bone fracture healing in rodents typically involve secondary healing and the formation of a mineralized callus. The shape of the callus formed and the density of the newly formed bone may vary substantially between timepoints and treatments. Whereas standard methodologies for quantifying parameters of intact cortical and trabecular bone are widely used and embedded in commercially available software, there is a lack of consensus on procedures for analyzing the healing callus. The purpose of this work is to describe a standardized protocol that quantitates bone volume fraction and callus mineral density in the healing callus. The protocol describes different parameters that should be considered during imaging and analysis, including sample alignment during imaging, the size of the volume of interest, and the number of slices that are contoured to define the callus.

Micro-computed tomography (&#181;CT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on &#181;CT procedures for analyzing the bone healing callus. This study provides a step-by-step &#181;CT protocol that allows the monitoring of fracture healing.

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed ...