This protocol is significant as it allows for time-specific targeted manipulation of mouse placentas using CRISPR. This will allow researchers to elucidate specific gene functions during mid to late pregnancy. Genetic manipulations causing overexpression within the mouse placenta are very uncommon.
The main advantage of this technique is it allows for a range of gene expression changes within the placenta. Do not get discouraged if preliminary terms have this technique are unsuccessful. As this technique is challenging, it requires quite a bit of time to practice to master.
To begin, place the anesthetized dam supine with a nose cone for maintaining anesthesia in the preparation area. Shave the abdomen of the dam thoroughly. To prevent corneal drying, place artificial tear gel over both eyes of the dam.
Then use sterile cotton tipped applicators to disinfect the surgical site with at least three alternating rounds of a povidone-iodine solution, followed by 70%ethanol with a final coat of povidone-iodine solution. After preparation, move the dam with the anesthesia nose cone to the designated surgery area. For the uterine laparotomy, place the dam supine on the operating table and secure the nose cone in place with tape.
Place a heating pad underneath an absorbent pad set to 45 degrees Celsius. After confirming the anesthesia depth via lack of toe pinch reflux, use forceps and scissors to make an approximate two-centimeter midline incision through the tented up skin. Then make a vertical incision through the peritoneum using sterile forceps, gently tented away from the uterine horns.
Massage both uterine horns by pressing on either side of the abdomen with fingers to guide them. Place the exposed uterus on top of sterile surgical drapes placed over the lower abdomen and keep it moist with drops of warm sterile saline as necessary. Once the uterus is exposed, select three pairs of placentas for manipulation.
Record the location of placental manipulations and the organization of the embryos within the uterine horns. For the placental injection of the control plasmid, load a sufficient quantity of the appropriate control plasmid for three injections using a calibrated micropipette. Perform all the injections between the decidua and junctional zone at a depth of approximately 0.5 millimeters laterally into the placenta.
After injection but immediately prior to electroporation, coat the places of contact with sterile saline, applying the saline precisely to the three sites on the uterine wall and the paddles with a dropper or syringe. Within two minutes of injection, perform electroporation using a pair of three-millimeter paddles attached to an electroporation machine. To ensure CRISPR incorporation efficiency and the viability of embryos, adjust the settings to 30 volts, 30-millisecond pulse, two pulses, 970-millisecond pulse off, and unipolar.
Gently press the electroporation paddles over the uterine walls on the lateral sides of the placenta. Place the anode paddle over the injection site and the cathode directly opposite. Press the pulse on the electroporation machine and wait for the two pulses to complete prior to removing the electroporation paddles.
Then proceed to placental injection of the experimental plasmid as demonstrated earlier. Perform electroporation of all three experimental injected placentas within two minutes of the injection as performed for the control group. Once the placental manipulation is complete, gently massage the uterine horns back inside the abdominal cavity using only fingers to directly manipulate the uterine horns.
Perform interrupted sutures on the peritoneum layer using 45 centimeters long coated and braided dissolvable sutures with a 13-millimeter, three-eighths circle needle alloy. After suturing the peritoneum layer, suture the skin with dissolvable sutures using the same type of dissolvable sutures used for the peritoneum. Once the suturing is complete, apply tissue adhesive to the sutures on the skin.
When the tissue adhesive has dried, turn off the vaporizer and transfer the dam from the surgery area to a supportive cage on its back. Placentas were injected with a general CRISPR Cas9 control plasmid and activation control CRISPR plasmid or an IGF-1 SAM activation plasmid. The representative post-necropsy images showed no noticeable damage or change in the phenotypic appearance in any treatment groups.
The survival rate of the untreated embryos in manipulated litters decreased to an average of 79.05%while there was a significant decrease in the survival of the manipulated embryos averaging 55.56%There was no significant difference in any group's placental weight. There was no significant difference in maternal weight gain immediately following surgery compared to sham surgeries. Many pregnant dams displayed a slight decrease or no change in weight the day after the procedure, likely due to disrupted eating during and briefly after the surgery.
Most dams showed increased weight on E14.5 but occasionally a decrease in weight was still observed. The qPCR showed a significant increase in IGF-1 expression in the IGF-1 overexpression placentas versus the control placentas. The ELIZA assessment of the E14.5 placentas showed a significant increase in IGF-1 protein levels in the IGF-1 overexpression placentas versus the control placentas.
Green autofluorescence demonstrated the sub regions of the placental maternal fetal interface with red Cas9 labeling in only the IGF-1 overexpression placenta, indicating successful CRISPR incorporation in all three sub regions of the placenta. The fluorescent in C2 hybridization labeling confirmed the incorporation of the IGF-1 activation plasmid with no migration into untreated placentas. The decidual and labyrinth zone could be identified surrounding the red junctional zone marked by Prl8a8 staining.
Remember to record the manipulation type and location of placentas and corresponding embryos. Recording the location of the cervix and embryos within the uterine horns is highly recommended. Following this procedure, analysis of the placenta, embryo, and dam can be performed.
This will allow for a greater understanding of genes placental specific roles in pregnancy, placental function, and embryonic development.
