This work was supported by funds from the Department of Animal and Avian Sciences to BT.

All animal experiments were approved by the Beltsville Area Animal Care and Use Comittees (BAACUC 11-015) in accordance with USDA Animal Care and Use Guidelines.
1. Anesthesia and Analgesia (Common for Both Surgical Procedures)
<ol>
	<li>Weigh the mouse and load the following anesthetics and analgesic in two 1 ml syringes with 27 G needles:
	<ol>
		<li>Ketamine (0.1 mg/g: 0.01 ml/g of a 10 mg/ml solution) and xylazine (0.01 mg/g: 0.005 ml/g of a 2 mg/ml solution).</li>
		<li>Buprenorphine (0.1 &#956;g/g: 0.01 ml of a 0.01 mg/ml solution).</li>
	</ol>
	</li>
	<li>Immobilize the mouse by picking up the scruff of its neck as close to the jaws as possible with the thumb and index fingers and holding the tail between the little and ring fingers.</li>
	<li>Inject Ketamine-Xylazine mixture intraperitoneally. In order to avoid puncturing internal organs, hold the mouse with its head slightly below the level of its hips (Figure&#160;1A)</li>
	<li>Inject Buprenorphine subcutaneously in the scruff of neck hold between the thumb and index fingers (Figure&#160;1B).</li>
	<li>Leave the mouse in the cage (clean and without any other animal) on a warm stage.</li>
	<li>Once unconscious, check for the absence of rear foot reflex (checked by toe pinch). Apply eye ointment to avoid dryness of the eye and to check for the absence of palpebral reflex (Figure&#160;1C).</li>
	<li>This protocol provides a surgical anesthesia plane for a minimum of 30 min, enough to perform the procedures described below (Protocols&#160;2 and 3). If longer times are required, an additional injection of ketamine + xylazine with half of the dosage described in 1.1.1 can be applied after 30 min. A change in the breathing pattern to a faster and irregular one indicates the loss of the proper anesthesia plane.</li>
</ol>
2. Vasectomy
<ol>
	<li>Use a male with a proven mating performance.</li>
	<li>Sterilize surgical instruments, clean the surfaces where surgery will be performed and wipe them with 70% ethanol.</li>
	<li>Perform anesthesia as previously detailed (Protocol 1), checking for loss of reflexes.</li>
	<li>Place the mouse on a warm stage, remove fur with electric clippers from the ventral area between two imaginary transversal lines placed 0.5 cm and 2.5 cm above the penis (Figure&#160;2A).</li>
	<li>Sanitize the shaved area by sequential wiping with 10% povidone iodine and 70% ethanol.</li>
	<li>Place the mouse in supine position with its tail towards the surgeon and cover with a sterile towel with a hole exposing the shaved area. Illuminate the surgical area.</li>
	<li>Perform a 10-15 mm longitudinal skin incision in the medial line of the abdomen, about 1 cm above the penis. Hold the skin with dressing serrated forceps and then cut with scissors (Figures&#160;2A and&#160;2B).</li>
	<li>Perform a 5-10 mm longitudinal incision in the linea alba. Hold the muscle with microdissecting serrated forceps and cut with scissors (Figure&#160;2C).</li>
	<li>Grab the testicular adipose pad of one side with micro dissecting serrated forceps and pull it to expose testis, vas deferens and epididymis. Vas deferens is located medial to the testis and it is a clearly distinguishable free tube (not attached to the testis wall like the epididymis) with a blood vessel running along one side (Figure&#160;2D).</li>
	<li>Holding the vas deferens with a micro dissecting serrated forceps, flame dressing forceps until they turn red (Figure&#160;2E), and then use them to cut and cauterize the vas deferens in two points at once (Figure&#160;2F). The cut should remove a portion of about 5 mm and leave two clearly separated cauterized ends (Figure&#160;2G).</li>
	<li>Move the testicle, epididymis and vas deferens back to the abdominal cavity.</li>
	<li>Proceed from step 9 in the other testicle.</li>
	<li>Suture the muscle with one or two horizontal mattress stitches made with 5/0 absorbable suture (Figure&#160;2H).</li>
	<li>Suture the skin with one or two wound clippers (Figure&#160;2I).</li>
	<li>Identify the vasectomised male (ear ring, finger tattoo&#8230;), move it the cage placed on a warm stage and observe until it recovers from anesthesia (conscious and maintain sternal recumbency). A 0.5-1 ml subcutaneous injection of warm saline solution improves recovery. Record the possible incidences occurring during vasectomy transfer, add antibiotic to the drinking water.</li>
	<li>Wound clips can be removed 10 days after vasectomy with a wound clipper remover or a pair of teeth forceps. The vasectomized male will be ready to mate 2 weeks after surgery.</li>
	<li>Test the infertility of the vasectomized male by mating with fertile females before using it to obtain recipients.</li>
</ol>
3. Utero-tubal Embryo Transfer
<ol>
	<li>Mouse morulae or blastocysts can be transferred by this technique to a pseudopregnant recipient female at 2.5 dpc.</li>
	<li>Prepare embryo manipulation glass pipette:
	<ol>
		<li>Polish the tips of the glass capillaries in order to avoid damaging the pipette holder.</li>
		<li>Soften a middle portion of the glass capillary by heating with a fine flame while slightly rotating the capillary with both hands synchronously. Once the capillary section becomes soft and malleable (light red color), withdraw it quickly from the flame and pull both ends to narrow its external diameter to 130-150 &#956;m.</li>
		<li>Wait for the glass to cool down and then cut it by lightly scoring the narrow part with a diamond-point pencil, abrasive stone or nail file and pulling from both sides. The break should be clean and perpendicular</li>
	</ol>
	</li>
	<li>Polish the tip by very quickly flaming, leaving a 100-130 &#956;m aperture. Pipettes can be stored for later use.</li>
	<li>Warm embryo manipulation media (CZBH or M2, see discussion).</li>
	<li>Sterilize surgical instruments, clean the surfaces where surgery will be performed and wipe them with 70% ethanol.</li>
	<li>Perform anesthesia as previously detailed (Protocol 1), checking for loss of reflexes.</li>
	<li>Keeping the mouse on a warm stage, remove fur with electric clippers from the dorsal area between the knees and the distal ribs (Figures 3A and&#160;3B).</li>
	<li>Sanitize the shaved area by sequential wiping with 10% povidone iodine and 70% ethanol.</li>
	<li>Move the embryos from the incubator to the prewarmed embryo manipulation media.</li>
	<li>Move the recipient to a warm stage under the stereomicroscope and place it in prone position laterally to the surgeon (with its head looking to the right or left side of the surgeon).</li>
	<li>Cover the area with a sterile towel with a hole exposing the shaved area and illuminate the surgical area.</li>
	<li>Perform a 1 cm transversal (vertical) incision in the skin in a spot located on the cranial &#8531; of the line between the last rib and the hips and the dorsal &#8531; of the line between the back and the abdomen (Figures 3A and&#160;3B). Hold the skin with dressing serrated forceps and cut with scissors (Figure&#160;3C).</li>
	<li>Once the skin has been cut, ovary (red/orange) or the adipose pad surrounding the ovary (white) can be visualized through the body wall. Perform a 0.3-0.5 cm transversal (vertical) incision in the body wall over the ovary or adipose pad in a spot where the incision does not cut any large blood vessel. Hold the muscle with micro dissecting serrated forceps and cut with scissors (Figure&#160;3D).</li>
	<li>Move the mouse to have its head facing towards the surgeon.</li>
	<li>Load the embryo manipulation pipette (Figure&#160;3E):
	<ol>
		<li>Allow CZBH media to ascend by capillarity through the narrow portion of the manipulation pipette until about 5 mm of the wider part.</li>
		<li>Take up a small air bubble (0.2-0.5 mm).</li>
		<li>Introduce the embryos (5-10) in a minimal amount of media (2-4 mm).</li>
		<li>Take up another small air bubble (0.2-0.5 mm) and a small amount of media (0.5-1 mm).</li>
		<li>Leave the glass pipette attached to the mouth aspirator holder or to the hand-operated device, ready for step 3.17.</li>
	</ol>
	</li>
	<li>Grab the adipose pad surrounding the ovary with micro dissecting serrated forceps and pull it towards the mouse head to expose the ovary, oviduct, and a small portion of the upper uterus out of the abdominal cavity (Figures 3F and&#160;3G).</li>
	<li>Holding the aspirator mouth piece in the mouth, ready to be used, grab the adipose pad with micro dissecting serrated forceps to move the oviduct and expose the utero-tubal junction (i.e. where the oviduct meets the uterus).</li>
	<li>Keeping the utero-tubal junction accessible, take slight curved micro dissection forceps with your left hand (if right handed) and place them just below the utero-tubal junction grabbing the oviduct about 2 mm above that portion.</li>
	<li>Holding the utero-tubal junction with the slight curved micro dissection forceps, puncture the oviduct section close to the forceps with a 27 G needle (Figure 3H).</li>
	<li>Insert the embryo manipulation pipette into the orifice performed with the needle and advance to the uterus through the utero-tubal junction (Figures 3I and&#160;3J). Once the pipette has passed the utero-tubal junction (Figure&#160;3K) it slides easily. Do not progress very far into the uterus to prevent endometrial damage (no more than 3 mm) and pipette blocking by the debris.</li>
	<li>Release the embryos into the uterus by gently blowing (Figure&#160;3L). Both air bubbles must pass through the uterus. Some of the media above the first bubble may also be released into the uterus, but avoid introducing more air, as it may impede implantation.</li>
	<li>Remove the pipette just after embryos have been released into the uterus.</li>
	<li>Move the oviduct and ovary back to the abdominal cavity by grabbing the adipose pad.</li>
	<li>Suture the muscle with a horizontal mattress stitch with 5/0 absorbable suture (Figure&#160;3M).</li>
	<li>Suture the skin with a wound clipper (Figure&#160;3N).</li>
	<li>Proceed from step 10 on the other side if required.</li>
	<li>Identify the recipient (ear ring, finger tattoo&#8230;), move it to its cage (placed on a warm stage) and observe until it recovers from anesthesia (conscious and maintain sternal recumbency).</li>
	<li>Annotate the possible incidences occurring during embryo transfer and add antibiotic to the drinking water. A 0.5-1 ml subcutaneous injection of warm saline solution improves recovery.</li>
	<li>Wound clips can be removed 10 days after embryo transfer with a wound clipper remover or two pairs of forceps (Figure&#160;3O). The recipient can be weighed on that day to assess pregnancy and estimate the number of pups. Provide nestling material to the recipient 15 days after embryo transfer.</li>
</ol>

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The authors declare no competing financial interests.

Vasectomy is a relatively straight forward surgical technique that does not involve major difficulties. When sanitizing with povidone iodine and ethanol make sure that the last wash (with ethanol) removes povidone iodine, as it may irritate the peritoneum. The access to vas deferens can also be achieved by the scrotum or performing a transversal incision in the abdomen8. Scrotal incision has been recommended to transversal abdominal incision due to the comparatively smaller incision needed and slightl...

Embryo transfer is probably the most frequent surgical procedure performed in the mouse model. This technique is essential to obtain offspring from embryos subjected to in vitro manipulation techniques and, therefore, constitutes a necessary step for the development of genetically modified models by pronuclear injection, lentiviral transduction, or chimera formation. Besides, the technique allows the study of the developmental effects of diverse insults occurring during preimplantation development. The use of artificial reproduction techniques1 or the exposure to abnormal concentrations of different substances or metabolites2 may affect embryo development resulting in implantation or placentation failures and long term effects in the offspring. A reliable and reproducible embryo transfer technique is crucial to test the possible negative effects of the experimental treatment on implantation and fetal development in a consistent manner.
Murine preimplantation embryos can be transferred to a recipient female either into the oviduct via the ampullae of 0.5 days post coitum (dpc) pseudopregnant recipients (oviduct transfer)3,4 or into the uterus of 2.5 dpc pseudopregnant recipient (uterine transfer)5,6 depending on their developmental stage. Embryos at the blastocyst stage, such as those used to generate chimeric mice by injection of embryonic or induced pluripotent stem cells, are usually transferred by uterine transfer. Blastocysts can also be transferred to the oviduct of a 0.5 dpc recipient, but it constitutes a less physiological test for developmental disruptors, because the embryo undergoes diapause and has 2 days to recover from the insult before implantation takes place. Uterine transfer involves puncturing the uterine wall with a narrow needle in order to generate an aperture that allows the access of an embryo manipulation pipette into the uterine lumen. Although this technique can yield good results, the survival to term (i.e. the percentage of embryos transferred that develop to a pup) is often low and unpredictable7,8.
The puncture of the uterine wall entails some detrimental side effects. First, myometrium is a highly vascularized tissue and its puncture often results in a small hemorrhage. Blood may block the embryo transfer pipette or invade the uterine lumen causing embryonic death and/or implantation failure. This is particularly relevant when embryos without zona are transferred, as the blood cells and debris can attach to the blastomeres. Second, the opening performed does not seal after the embryos have been transferred, so they can flow back through the orifice and be expelled to the abdominal cavity when a too large volume has been introduce into the uterus. The utero-tubal embryo transfer described herein take advantage of the utero-tubal junction to deliver the embryos into the uterus without the need of puncturing the uterine wall and thereby avoiding its adverse consequences9.
The pseudopregnant recipient females used for embryo transfer are obtained by natural mating with vasectomized males8. The seminal secretions produced by a sterile male are required for the uterus to become receptive to the transferred embryos. To obtain a recipient, a maximum of 2 females of 8 weeks to 6 months of age are placed with a vasectomized male in the afternoon. The following morning, females are checked for the presence of a vaginal copulation plug, a clump of coagulated proteins from the male seminal fluid. As mating usually occurs during midnight, the day of vaginal plug detection is considered to be 0.5 dpc. Although vasectomized males can be purchased from some vendors, the surgical procedure described herein is relatively easy and does not require any additional instruments than required for embryo transfer.

<table><tbody><tr><td>Ketamine</td><td>VEDCO</td><td>Ketaved ANADA 200-257</td><td>To be ordered by a licensed veterinarian.</td></tr><tr><td>Xylazine</td><td>Lloyd Laboratories</td><td>Anased NADA #139-236</td><td>To be ordered by a licensed veterinarian.</td></tr><tr><td>Buprenorphine</td><td>Generic</td><td>NDC 400-42-010-01</td><td>To be ordered by a licensed veterinarian.</td></tr><tr><td>Eye ointment</td><td>Novartis</td><td>Genteal</td><td></td></tr><tr><td>Antibiotic</td><td>Pfizer</td><td>Clavamox NADA #55-101.</td><td>Added to drinking water (0.3 mg/ml of amoxicillin trihydrate and 0.075 mg/ml of clavulanate potassium). Add 1.5 ml of the reconstituted 15 ml bottle to 250 ml of water.</td></tr><tr><td>Dressing serrated forceps</td><td>ROBOZ</td><td>RS-8120</td><td>Any medium size surgical-grade steel straight forceps will work.</td></tr><tr><td>Microdissecting serrated forceps</td><td>ROBOZ</td><td>RS-5137</td><td>These ones are curved at a 90&amp;ordm; angle. Straight forceps can be used if preferred.</td></tr><tr><td>Slight curved micro dissection forceps</td><td>ROBOZ</td><td>RS-5136</td><td>This model is particularly useful to hold the oviduct.</td></tr><tr><td>Scissors</td><td>ROBOZ</td><td>RS-5880</td><td>Any regular surgical grade steel small straight scissors will work.</td></tr><tr><td>27 G needles</td><td>Beckton-Dickinson</td><td>305136</td><td>Smaller needles (30 G) can be also used. 25 G may be a bit too big.</td></tr><tr><td>Clip applier</td><td>MiKRon</td><td>42763</td><td></td></tr><tr><td>9 mm Clips</td><td>MiKRon</td><td>427631</td><td></td></tr><tr><td>Clip remover</td><td>MiKRon</td><td>7637</td><td>Two pairs of teeth forceps (ROBOZ RS-8160) can be used instead.</td></tr><tr><td>Suture needle holder</td><td>ROBOZ</td><td>RS-7820</td><td></td></tr><tr><td>Suture</td><td>Dowist Gell</td><td>5-0 Dexon S 7204-21</td><td>Can be substituted for any 4-0 to 6-0 absorbable suture with a narrow curved needle.</td></tr><tr><td>Glass capillaries</td><td>VWR</td><td>100 ul calibrated pipettes 53432-921</td><td>It includes a mouth aspirator system that only requires to attach a 0.22 &amp;#181;m filter in the tubing to be ready to use. More information can be obtained in Nagy &lt;em&gt;et al&lt;/em&gt;.&lt;sup&gt;8&lt;/sup&gt;</td></tr><tr><td>Burner</td><td>KISAG AG</td><td>Typ 2002</td><td>Gas operated burner, can be charged with Kigas (CH-4512, from the same vendor). Alcohol burners may be also used, but gas provides a higher temperature and this burner provides a small and precise flame.</td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>MZFLIII</td><td>This is an expensive estereomicroscope with fluorescence, that can be also used for other purposes. There are cheaper options such as Leica MZ8 or Nikon SMZ-10 or SMZ-2B, to name a few. It is better to use two stereomicroscopes, one for handling the embryos (which does not need to be a very nice one) and another one for the recipient. The one used for the recipient should display a long distance from the stage plate to the objective lense, in order to be able to focus 3-4 cm above the stage plate (where the oviduct will be placed) and still leave some room for the surgeon; most of the stereomicroscopes can do this, but some cannot.&amp;nbsp;</td></tr><tr><td>Fiber optics ilumination</td><td>Dolan Jenner</td><td>Fiber lite</td><td>To iluminate the surgical area. There are different systems available.</td></tr><tr><td>Warm stages</td><td>American scope</td><td>&lt;a href=&quot;https://exch.mail.umd.edu/owa/redir.aspx?C=1qTJKejamUSfudeYG_MlZHvTY3dsJM9Iaqr2SnwGD4ECWGzB8e8Rpg--_L7u0vJmyatXQJzGQ0k.&amp;amp;URL=http%3a%2f%2fstore.amscope.com%2ftcs-100.html&quot; target=&quot;_blank&quot;&gt;http://store.amscope.com/tcs-100.html&lt;/a&gt;</td><td>These can be placed over the stage plate of the stereomicroscope. Some modifications (inserting a stick to level the stage) may be needed if it is too short for the stereomicroscope. A big warm stage can be used for warming the cage if it is available. If not, a regular heating pad can be used, but temperature must be checked.</td></tr><tr><td>Culture dishes for embryo manipulation</td><td>Falcon</td><td>353001</td><td>351008 may be also used; they made narrower drops.</td></tr></tbody></table>

utero-tubal embryo transfer and vasectomy in the mouse model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Utero-tubal embryo transfer provides a mean to transfer embryos to the uterus avoiding some of the complications associated to the uterine embryo transfer2,9,10. In Table 1 we show some representative result we have obtained transferring CD1 blastocysts subjected to different kinds of manipulations to CD1 recipients following the protocol described. The survival to term (% of embryos resulting in a pup) or survival to E15 (in the case of lentivirus exposed) is similar between embryos simply cu...

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Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

utero-tubal-embryo-transfer-and-vasectomy-in-the-mouse-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

47.7K Views.  United States Department of Agriculture. Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

Video: Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Medicine

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Neuroscience

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3 and later transferred to the mouse4. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8, hormones9,10 and environmental factors11,12 affect endometriosis as well as the effects of endometriosis on fertility13 and pain14.

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

Genetics

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

Developmental Biology

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation and placenta formation and enable ongoing pregnancy. The limitations to pregnancy success caused by embryonic defects are well known and have been largely overcome in recent decades with the rise of in vitro fertilization (IVF) and assisted reproductive technologies. As yet, however, the field has not overcome the limitations caused by an inadequately receptive endometrium, thus resulting in stagnating IVF success rates. Ovarian and endometrial functions are closely intertwined, as hormones produced by the ovary are responsible for the endometrium's menstrual cyclicity. As such, when using rodent models of pregnancy, it can be difficult to ascertain whether an observed result is due to an ovarian or uterine deficit. To overcome this, an ovariectomized mouse model was developed with embryo transfer or artificial decidualization to allow the study of uterine-specific contributions to pregnancy. This article will provide instructions on how to perform ovariectomy and offer insights into various techniques for supplying exogenous hormones to support successful artificial decidualization or pregnancy following embryo transfer from healthy donors. These techniques include subcutaneous injection, slow-release pellets, and osmotic mini pumps. The key advantages and disadvantages of each method will be discussed, enabling researchers to choose the best study design for their specific research question.

Pregnancy establishment is a dynamic process involving complex embryo and uterine crosstalk. The precise contributions of the maternal uterine environment to these processes remain an active area of investigation. Here, detailed protocols are provided to aid in designing in vivo animal models to address these research questions.

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation ...

Murine Round Spermatid Injection Procedure to Study Embryonic Development

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

ICSI-ET Mouse Model to Study Long-Term Impacts on Glucose Metabolism

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life roughly equivalent to 40 human years. Intracytoplasmic sperm injection (ICSI) is a commonly used assisted reproductive technology in clinical practice. However, given its relatively recent emergence about 30 years ago, the long-term effects of this technique on human development remain unclear. In this study, we established the ICSI combined with embryo transfer (ET) method using a mouse model. The results demonstrated that normal mouse sperm, after undergoing in vitro culture and subsequent ICSI, exhibited a fertilization rate of 89.57% and a two-cell rate of 87.38%. Following ET, the birth rate of offspring was approximately 42.50%. Furthermore, as the mice aged, fluctuations in glucose metabolism levels were observed, which may be associated with the application of the ICSI technique. These findings signify that the mouse ICSI-ET technique provides a valuable platform for evaluating the impact of sperm abnormalities on embryo development and their long-term effects on offspring health, particularly concerning glucose metabolism. This study provides important insights for further research on the potential effects of the ICSI technique on human development, emphasizing the necessity for in-depth investigation into the long-term implications of this technology.

Author Spotlight: Exploring the Long-Term Health Impacts of Intracytoplasmic Sperm Injection on Offspring

Here, we present a protocol to establish an intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) mouse model, allowing us to observe age-related changes in glucose metabolism that may be attributed to ICSI, providing insights into its potential long-term impacts on human development.

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life ...

Cervical Manipulation Induced Pseudopregnancy in Mice for Assisted Reproduction

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.

Author Spotlight: Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination  

The cervical manipulation method presented can induce pseudopregnancy in mice without the need for breeding females with vasectomized males. The induction of pseudopregnancy is required for the success of non-surgical embryo transfer and non-surgical artificial insemination, both of which are also presented.

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;female recipient mice must be induced into a pseudopregnant ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

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A Mouse Model of in Utero Transplantation

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

Mouse Round Spermatid Injection

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination