We would like to thank the Equine Veterinary Hospital and the Ruminants Veterinary Hospital of the College of Veterinary Medicine, University of Sao Paulo (FMVZ/USP), Sao Paulo, Brazil, FAPESP (grant 2022/11459-3, Sao Paulo Research Foundation), EMS Pharma, CNPq (grant 405254/2022-9), and &#193;gua Branca pig farming, Itu, Sao Paulo, Brazil.

This study was approved by the Ethics Committee on Animal Use in Research of the College of Veterinary Medicine and Animal Science of the University of S&#227;o Paulo, protocol number 6088030523. Nine seven-month-old gilts from the &#193;gua Branca nucleus pig farming, located in Itu city, S&#227;o Paulo state, Brazil, were used right after the second estrus detection9. The details of the reagents and the equipment used in the study are listed in the Table of Materials.
1. Animal preparation
<ol>
	<li>Fast the animals for 12 h and restrict water access for 4 h before the surgical procedure.</li>
	<li>Using a 40 mm x 12 mm needle, draw up 10 mg/kg of ketamine and 0.2 mg/kg of midazolam in the same 50 mL syringe. Attach the syringe to an IV administration set to facilitate medication administration, maintaining a safe distance from the animal and allowing it to move freely.</li>
	<li>Administer the pre-anesthetic medication via intramuscular injection into the trapezius muscle, caudal to the external ear canal.</li>
	<li>Cannulate the auricular vein with a 20 G catheter and connect it to a macrodrip administration set for intraoperative fluid therapy with 0.9% NaCl solution at a rate of 10 mL/kg/h.</li>
	<li>Induce anesthesia with 4 mg/kg of propofol intravenously (following institutionally approved protocols).</li>
	<li>Perform orotracheal intubation of the animal using a laryngoscope and a size 9 orotracheal tube with a cuff10.</li>
	<li>Maintain anesthesia with 1.5% to 2% isoflurane inhalation. Additionally, monitor anesthesia by evaluating physiological parameters such as heart rate, which should be maintained between 100 and 120 beats per minute, oxygen saturation, which should be kept between 99% and 100%, muscle relaxation, absence of movement, and reflexes evaluation. 
	NOTE: Body temperature should also be monitored and controlled, staying between 37.5-38.5 &#176;C. When necessary, in addition to isoflurane, propofol can be used to maintain control of the parameters. Fentanyl hydrochloride should be administered as an analgesic at a dose of 0.02 mg/kg every 20 min. All parameters should be recorded every 5 min.</li>
	<li>Administer perioperative analgesia with a single dose of 0.002 mg/kg of fentanyl intravenously and 4 mg/kg of tramadol hydrochloride intramuscularly into the gluteal or trapezius muscle.</li>
	<li>Place cardiac electrodes on the thoracic region for intraoperative monitoring of heart rate. Record heart rate, rectal temperature, and oxygen saturation every 5 min.</li>
</ol>
2. Surgical procedure
<ol>
	<li>Position the gilt on the surgical table in dorsal recumbency.</li>
	<li>Cover the animal&#39;s legs with procedural gloves to minimize contamination of the surgical field.</li>
	<li>Perform ventral caudal abdominal hair removal between the penultimate pair of mammary glands using a surgical clipper.</li>
	<li>Perform preoperative skin antisepsis with a chlorhexidine-impregnated antiseptic brush, making circular movements from the umbilical scar region to the last pair of mammary glands. Remove chlorhexidine with a clean, moistened compress.</li>
	<li>Ensure that the surgeon and the assistant perform surgical antisepsis and wear sterile surgical gowns and gloves for surgery. 
	NOTE: The animal should be prepared for surgery in a preparation area separate from the surgical site.</li>
	<li>Prepare the surgical table with the appropriate and previously sterilized instruments listed in the Table of Materials.</li>
	<li>Perform final surgical field antisepsis using sterile gauze and chlorhexidine antiseptic, cleaning the region in a fishbone pattern, starting at the incision site and expanding laterally.</li>
	<li>Repeat this procedure three times with chlorhexidine antiseptic, and then repeat the procedure with alcoholic chlorhexidine.</li>
	<li>Completely cover the animal with a sterile surgical drape, and using scissors, create a rectangular opening in the surgical drape, approximately 10 x 20 cm&#178;, if the drape is not fenestrated.</li>
	<li>Position the surgical drape over the penultimate pair of inguinal mammary glands. Place 4 Backhaus clamps to secure the surgical drape over the animal&#39;s skin.</li>
	<li>Incise the skin with a scalpel, creating an incision approximately 10 cm in length.</li>
	<li>Achieve hemostasis of subcutaneous vessels with hemostatic forceps and/or absorbable suture size 2.</li>
	<li>Dissect the subcutaneous tissue with fingers or with the aid of fine-pointed scissors, deepening the incision to reach the linea alba in the abdominal musculature. 
	NOTE: Palpate the midline of the abdomen to feel the linea alba as a more rigid and fibrous area; observe the separation of the rectus abdominis muscles and note the change in tissue consistency while dissecting to identify the linea alba accurately11.</li>
	<li>With Allis forceps, grasp the musculature and lift it to make a stab incision in the linea alba with a scalpel.</li>
	<li>Insert the index finger into the incision to assess the presence of adhesions. Extend the incision over the linea alba caudally and cranially with blunt/blunt or fine/blunt scissors carefully to avoid injuring other organs and underlying structures.</li>
	<li>Make an incision of sufficient length so that the surgeon&#39;s one hand can be inserted into the abdominal cavity to locate and exteriorize the uterine horns and/or ovaries. A Gosset retractor may be used to facilitate entry into the abdominal cavity and suturing of the musculature in a single layer at the end of the procedure.</li>
	<li>If one of the uterine horns is located before the ovaries, gently follow it until reaching the tip of the uterine horn and the ipsilateral ovary. During this procedure, assess the presence of fluid inside the uterus, which may compromise gestation.</li>
	<li>After locating the ovary, gently pull it and carefully expose it. Assess the presence of pre-ovulatory follicles, hemorrhagic corpora lutea, cyclic corpora lutea, and corpus albicans to evaluate if the gilt&#39;s synchronization is as expected.</li>
	<li>Gently remove the fimbriae of the uterine tube covering the ovary, evert its mucosa, and locate the ostium of the uterine tube.&#160; 
	NOTE:&#160;If the surgeon finds it difficult to identify the ostium of the fimbriae, a pocket on the ampulla of the oviduct can be done with a round-bodied suture needle, avoiding puncturing blood vessels, to insert the Tomcat catheter containing the embryos.</li>
	<li>Carefully insert a Tomcat catheter containing the embryos into the ostium of the fimbriae until reaching the ampulla region of the uterine tube.</li>
	<li>Attach a 1 mL syringe to the Tomcat catheter to push the embryo-containing fluid into the uterine tube. Cover the ovary with the fimbriae and return it to the abdominal cavity.</li>
	<li>Locate the contralateral uterine horn and ovary and repeat steps 2.17-2.21.</li>
	<li>Reposition the structures inside the abdominal cavity and add 1 L of saline solution or Ringer&#39;s lactate solution warmed (39 &#176;C) to prevent adhesions before initiating abdominorraphy.</li>
	<li>Suture the abdominal musculature with absorbable suture size 2 in &#34;X&#34; stitches (Sultan) interrupted.</li>
	<li>Approximate the subcutaneous tissue with a continuous mattress suture using absorbable suture size 2.</li>
	<li>Close the skin with size 2 nylon suture in interrupted simple stitches, surgical staples, or ethyl-cyanoacrylate adhesive. In this procedure, we opted for the use of adhesive.</li>
	<li>Clean the surgical wound with gauze soaked in hydrogen peroxide and dry with a compress to enhance the fixation of the surgical adhesive on the surgical wound. Apply rifamycin or Pearson&#39;s ointment, cover with gauze, and finally, apply surgical adhesive. 
	NOTE: The wound should remain closed with a dressing for 5 days.</li>
</ol>
3. Postoperative care
<ol>
	<li>Evaluate the animals for signs of characteristic clinical pain for at least the next 3 consecutive days, according to the scale proposed by Luna et al.12. 
	NOTE: This scale is based on different behaviors exhibited by pigs when experiencing different levels of pain, such as posture, interaction and interest in the environment, activity, appetite, and attention to the affected area. The score ranges from 0 to 3, depending on the type of behavior exhibited, for each parameter evaluated. At the end of the evaluation, the sum of all scores reflects the intensity of pain experienced by the animal, where 0 represents no pain, and 18 represents very intense pain12.</li>
	<li>Administer a single dose of 15 mg/kg of amoxicillin and 0.4 mg/kg of 2% meloxicam both intramuscularly (in the gluteal or cervical muscle) once a day for 3 days as postoperative care to prevent pain, inflammatory processes, and infection. 
	NOTE: If the pain score is equal to or greater than 6 on the pain scale used (score from 0 to 18), administer 5,000 mg/animal of sodium dipyrone intramuscularly (in the gluteal or cervical muscle) once a day in addition to the anti-inflammatory and antibiotic. Alternatively, perform analgesia as outlined in the local IACUC guidelines.</li>
	<li>On the 6th day after surgery, remove the surgical adhesive for wound cleaning with gauze soaked in 2% alcoholic chlorhexidine solution and apply topical Pearson&#39;s ointment until the 10th day, when suture removal or surgical staples removal, if used, is recommended.</li>
</ol>
4. Ultrasonographic gestational diagnosis of pregnancy
<ol>
	<li>Perform transabdominal pregnancy ultrasonographic evaluation with an ultrasound device (frequency range 4.5 to 8.5 MHz) 30 days after embryo transfer surgery.</li>
	<li>Apply ultrasound gel to the probe and position it near the inguinal abdominal region, facing medially. 
	NOTE: Visualization of rounded anechoic embryonic vesicles, due to fluid in the uterine lumen, indicates gestation. Non-visualization of intrauterine fluid, or&#160;presence of intrauterine fluid without delineation of embryonic vesicles, suggests the absence of gestation.</li>
</ol>
5. Euthanasia of the animals
NOTE: &#160;Euthanize the gilts that did not present fetuses of size and appearance corresponding to gestational age on the ultrasonographic examination.
<ol>
	<li>Using a 40 mm x 12 mm needle, draw up 15 mg/kg of ketamine and 2 mg/kg of midazolam in the same 50 mL syringe attached to an intravenous administration set, and administer intramuscularly into the trapezius muscle in the cervical region caudal to the external ear canal, as preanesthetic medication.</li>
	<li>Cannulate the auricular vein with a 20 G catheter and induce anesthesia with 4 mg/kg of propofol intravenously in a bolus until bradycardia occurs (following institutionally approved protocols).</li>
	<li>Subsequently, administer 10 to 20 mL of 19.1% potassium chloride intravenously. Confirm cardiopulmonary arrest by observing the absence of respiratory movements, pale mucous membranes, loss of corneal reflex, and absence of heartbeats by placing a stethoscope over the cardiac region.</li>
	<li>Collect any type of intrauterine content (embryonic tissue, liquid, or mucous secretions) from necropsied females13 and forward it for microbiological analysis to detect possible pathogens and also for genotypic characterization of the clones (if needed). Visually check the presence or absence of corpora lutea, ovarian cysts, abnormal secretions, and the occurrence of adhesions in the uterus, ovaries, and oviducts14.</li>
</ol>

Optimizing Oviductal Embryo Transfer in Cloned Pigs

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None of the authors disclose any conflict of interest

The surgical method described has been previously performed by other research groups working with the production of cloned pigs or genetically modified cloned pigs, with reports of births after the implementation of this technique15,16,17,18,19,20. The pregnancy and birth rates of SCNT embryos produced from various cell lines...

Pigs are an excellent experimental model for biomedical research due to their anatomical, physiological, and genetic similarities to humans1. These animals have often been used in research related to xenotransplantation, with the intention of producing organs, cells, or tissues that promote a low risk of rejection when transplanted into humans. Xenotransplantation research aims to increase the organ supply for human transplantation, thus reducing the waiting list of patients2.
The production of pigs for xenotransplantation involves several steps, including the production of clones from genetically edited swine cells. After the in vitro production of genetically modified cloned embryos, the embryos are transferred to the reproductive system of a sow with a synchronized estrous cycle to prepare the uterine physiology for the reception and gestation of the new conceptus3.
Embryo transfer in pigs can be performed by non-invasive or invasive methods4. Among the non-invasive methods is transcervical transfer, which does not require any surgical intervention. However, this method is restricted to transferring embryos at later development stages (i.e., morula or blastocyst stages) and does not allow for the precise determination of the catheter insertion site or embryo deposition5. Laparoscopy and laparotomy are considered invasive methods of embryo transfer. Laparoscopy is less invasive but requires specific and costly equipment, and its efficiency varies considerably (from less than 20% to over 80%) due to various factors such as difficulty in manipulating reproductive structures and the type of catheter used4,6. Therefore, transfer via laparoscopy is still less efficient compared to surgical transfer methods via laparotomy4.
Embryo transfer surgery in sows via laparotomy is a relatively simple and quick procedure, typically taking about 30 min. However, it should be performed in a surgical center equipped with inhalation anesthesia apparatus and a specialized team. For commercial pig strains (such as Landrace, Large White, or their crossbreeds), special equipment like hoists for lifting gilts and a wide, sturdy surgical table are necessary due to the animals&#39; considerable weight (around 130-150 kg).
For the surgery to be successful, the embryos must be evaluated for their stage of development beforehand. Embryos of up to 4 cells are recommended to be transferred into the uterine tube. Embryos at stages beyond 4 cells, such as morulas and blastocysts, should be transferred to the uterine horn7,8.
Although research groups worldwide are performing the production and surgical transfer of genetically modified cloned pig embryos, there are still no well-defined protocols demonstrating this procedure through videos. This approach is crucial for the success of gestation, as the technique requires precise deposition of embryos in the exact location of the oviduct, involving the localization of the tubal ostium and the introduction of the pipette containing the embryos. This technique can be better understood through explanatory videos of the entire procedure. Therefore, this article aims to demonstrate the laparotomy surgery for transferring cloned embryos to the oviduct of gilts, an essential prerequisite for the future production of genetically modified pigs to be used for xenotransplantation or other related purposes.

<table><tbody><tr><td>0.25 mL straw</td><td>generic</td><td>-</td><td>Surgical material</td></tr><tr><td>1 mL syringe</td><td>Descarpack</td><td>341001</td><td>Surgical material</td></tr><tr><td>10 mL syringe</td><td>Descarpack</td><td>324601</td><td>Surgical material</td></tr><tr><td>20 mL syringe</td><td>Descarpack</td><td>324801</td><td>Surgical material</td></tr><tr><td>3 mL syringe</td><td>Descarpack</td><td>324201</td><td>Surgical material</td></tr><tr><td>5 mL syringe</td><td>Descarpack</td><td>324401</td><td>Surgical material</td></tr><tr><td>60 mL syringe</td><td>Descarpack</td><td>323201</td><td>Surgical material</td></tr><tr><td>9 mm endotracheal tube</td><td>Rusch</td><td>112482-000090</td><td>Surgical material</td></tr><tr><td>Allis forceps</td><td>generic</td><td>-</td><td>Surgical instrument</td></tr><tr><td>Amox LA</td><td>JA Sa&amp;uacute;de Animal</td><td>MAPA registration: 8.781/2004</td><td>Pharmaceutical drug</td></tr><tr><td>Bakhaus forceps</td><td>generic</td><td>-</td><td>Surgical instrument</td></tr><tr><td>Catheter 20G</td><td>Descarpack</td><td>362401</td><td>Catheter for intravenous access</td></tr><tr><td>Cetamin</td><td>Agener Uni&amp;atilde;o</td><td>MAPA registration: SP-000292-5.000011&amp;nbsp;</td><td>Anesthetic</td></tr><tr><td>Conductive clinical gel</td><td>RMC</td><td>ANVISA registration: 80122200013</td><td>Surgical material</td></tr><tr><td>Dipyrone D-500</td><td>Zoetis</td><td>MAPA registration: SP0000728-46</td><td>Pharmaceutical drug</td></tr><tr><td>Disposable scalpel n. 22</td><td>Wiltex</td><td>ANVISA registration: 10150470565</td><td>Surgical instrument</td></tr><tr><td>Disposable sterile sponge-brush</td><td>Rioquimica</td><td>7.89778E+12</td><td>Surgical asepsis</td></tr><tr><td>Easy-Scan:Go</td><td>IMV</td><td>ESCG01</td><td>Ultrasound</td></tr><tr><td>Endozime AW Plus</td><td>Ruhof</td><td>34514</td><td>Detergent for surgical instruments</td></tr><tr><td>Fentanil (Fentanest)</td><td>Crist&amp;aacute;lia</td><td>ANVISA registration: 1029800810159</td><td>Anesthetic</td></tr><tr><td>Gosset retractor</td><td>generic</td><td>-</td><td>Surgical instrument</td></tr><tr><td>Halstead-mosquito hemostatic forceps</td><td>generic</td><td>-</td><td>Surgical instrument</td></tr><tr><td>Healing ointment - 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embryo transfer surgery via laparotomy in gilts

This protocol aims to demonstrate the surgical technique for transferring cloned pig embryos to the oviduct, a method widely used in the production of genetically modified pigs for biomedical research. Nine gilts underwent hormonal synchronization and laparotomy for the transfer of cloned embryos produced by somatic cell nuclear transfer (SCNT) at stages of up to 4 cells on day 2 to the oviduct. Gestational diagnosis was conducted via ultrasound examination 30 days post-transfer surgery. Six out of the nine operated gilts exhibited signs of pregnancy on ultrasound examination. However, as there was no progression in fetal development as assessed by echography, the gilts underwent necropsy at 60 days for the collection of biological material and assessment of the reproductive system. Adhesions were observed in the uterine horns, ovaries, and oviducts. From the uterine lumen of two of the euthanized gilts, one and four embryonic structures with gestational ages ranging between 12 to 20 days were obtained. Despite the absence of live piglets, likely attributed to the low-efficiency rate of transferring cloned pig embryos, which is influenced by various factors, including the number and quality of transferred embryos, the presented surgical technique proved to be rapid and safe.

This article aims to demonstrate the laparotomy surgery for transferring cloned embryos to the oviduct of gilts. All animals remained in an adequate anesthetic plane, without any intraoperative incidents or complications during anesthesia recovery. The gilts took, on average, 2-3 h to stand up after the surgery ended.
All surgical procedures lasted, on average, 44 min. Nine gilts underwent surgery, with an average of 185 cloned embryos transferred per gilt, totaling 1,664 embryos transferred b...

Author Spotlight: Surgical Methods and Outcomes in Oviductal Cloned Pig Embryo Transfers

This protocol describes the application of the surgical technique used to transfer cloned pig embryos via laparotomy in gilts.

Embryo Transfer Surgery via Laparotomy in Gilts

This protocol aims to demonstrate the surgical technique for transferring cloned pig embryos to the oviduct, a method widely used in the production of ...

embryo-transfer-surgery-via-laparotomy-in-gilts

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JoVE Journal

Medicine

584 Views.  Universidade de São Paulo. This protocol describes the application of the surgical technique used to transfer cloned pig embryos via laparotomy in gilts.

Video: Author Spotlight: Surgical Methods and Outcomes in Oviductal Cloned Pig Embryo Transfers

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 ...

Developmental Biology

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Neuroscience

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

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 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 ...

Biology

Intubation, Central Venous Catheter, and Arterial Line Placement in Swine for Translational Research in Abdominal Transplantation Surgery

Translational surgical research models in swine are crucial for developing safe preclinical protocols. However, the success of the experimental surgeries does not solely rely on the research team's surgical skills; perioperative care and management procedures, like intubation, central venous line, and arterial line placement, are necessary and of the utmost importance for favorable experiment results. As it is uncommon for research teams to have anesthesiologists or any other staff other than the surgical team, the surgical team involved in translational research must acquire and/or develop the skills to perform the perioperative care. The purpose of this paper is to show the techniques of intubation, central venous catheter, and arterial line placement used and perfected at the Toronto Organ Preservation Laboratory over the last 10 years, to be used as a reference for future researchers joining either this team or any other lab performing translational research protocols in swine and/or abdominal transplantation.

To obtain the best possible results, surgeons performing translational research experiments should be proficient at intubation and vascular line placement. This paper describes the techniques used by the Toronto Organ Preservation Laboratory to perform these procedures.

Translational surgical research models in swine are crucial for developing safe preclinical protocols. However, the success of the experimental surgeries ...

Surgical Approach, Challenges, and Resolutions for Uterus Transplantation in Rats

Uterine transplantation (UTx) is a new approach for treating women with absolute uterine factor infertility (AUFI). An estimated 3%-5% of women suffer from AUFI. These women were deprived of the option to have children until the advent of UTx. The clinical application of UTx was driven by experimental studies in animals, and the first successful UTx was achieved in rats. Given their physiological, immunological, genetic, and reproductive characteristics, rats are a suitable model system for such transplants. In particular, their short gestation period is a clear advantage, as the usual endpoint of experimental UTx is successful pregnancy with live birth. The biggest challenge for rat models remains the small anatomy, which requires advanced microsurgical skills and experience. Although UTx has led to pregnancy in the clinic, the procedure is not established and requires continuous experimental optimization. Here, a detailed protocol is presented, including essential troubleshooting for rat UTx, which is expected to make the entire procedure easier to grasp for those without experience in this type of microsurgery.

The present protocol describes all the essential steps for successful uterine transplantation (UTx) in rats. The rat model has proven suitable to promote the clinical implementation of UTx; however, rat UTx is a highly complex procedure requiring careful instructions.

Uterine transplantation (UTx) is a new approach for treating women with absolute uterine factor infertility (AUFI). An estimated 3%-5% of women suffer ...

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants with congenital diaphragmatic hernia (CDH) also have lung hypoplasia due to occupation of the chest cavity by the stomach and bowel and, in the most severe cases, the liver. Balloon tracheal occlusion reduces the severity of lung hypoplasia in fetuses with CDH but increases the risk of premature birth. Understanding the optimal occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improving in utero corrective treatments for CDH. The study reports a new method for continuous measurement of the intratracheal and amniotic pressures in an unoccluded and occluded fetal lamb surgical model of CDH. Time-pregnant Merino ewes underwent two recovery hysterotomies: the first at ~80 days of gestation to create the CDH, and the second at ~101 days of gestation to occlude the fetal trachea and implant an intratracheal and amniotic pressure measurement device. Lambs were delivered at ~142 days, and the pressure measurement device was removed and cleaned. The data were downloaded and filtered using a 6 h window. Transrespiratory pressure was calculated.

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

Optimization of occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improve in utero corrective treatments for congenital diaphragmatic hernia (CDH). This study reports a new method for continuous measurement of the tracheal pressure in an occluded and unoccluded fetal lamb surgical model of CDH.

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants ...

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...

Embryo Transfer Into Rabbit: A Procedure for Laparoscopy-Guided Transfer of Embryos Into Oviduct of Ovulating Recipient Female Rabbit Model

Source: Garcia-Dominguez, X. et al., <a href="https://www.jove.com/v/58055">Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model.</a> J. Vis. Exp. (2019)
This video describes the technique of transferring embryos into the oviduct of a recipient female rabbit via Laparoscopy. It allows embryo remodeling in rabbits during its migration through the oviduct, eventually helping in successful implantation. This technique can also be used for stored embryos with good efficiency.

Source: Garcia-Dominguez, X. et al., Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model. J. Vis. Exp. ...

Embryo Transfer Surgery via Laparotomy in Gilts

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Chapters in this video

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

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

Intubation, Central Venous Catheter, and Arterial Line Placement in Swine for Translational Research in Abdominal Transplantation Surgery

Surgical Approach, Challenges, and Resolutions for Uterus Transplantation in Rats

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

Embryo Transfer Into Rabbit: A Procedure for Laparoscopy-Guided Transfer of Embryos Into Oviduct of Ovulating Recipient Female Rabbit Model