Mice transgenic for antigen specific T cell receptors have been indispensable in the study of T cell development and function to generate mouse models with predefined T-cell receptor specificities using somatic cell nuclear transfer or SCNT. A mouse is infected with a pathogen of interest. T-cells are isolated from the mouse and then purified by fluorescent activated cell sorting.
The purified T-cell are then used for somatic cell nuclear transfer. After the embryos reach the blasts stage, embryonic stem cells are isolated. These cells are then injected into blast assist, which are then transferred to pseudo pregnant mice.
The resulting pups express a pronounced increase in the T-cell population of interest, which can be validated by PAX analysis. Hello, my name is Okta AK and I'm a postoc here at the White Institute for Biomedical Research. Today I'm going to show you how to perform somatic CLE transfer blast assist injections as well as embr transfer.
The main advantage of somatic transfer is that mouse models can be generated, which express their T-cell receptor from the endogenous locus, and therefore under control of the endogenous promoter and that mouse models can be generated very fast. Somatic send liquid transfer can be used as a tool to generate mouse models with predefined T-cell receptor or B-cell receptor specificities. This is of special interest for people working in the field such as autoimmunity, infectious disease, and cancer.
To isolate T-cell specific for a pathogen such as Toxoplasma Gandhi, miser infected by intraperitoneal injection at the peak of the immune response, the spleen is isolated from the euthanized mouse and homogenized donor cells are then isolated and incubated with fluorescently labeled antibodies and the appropriate tetramer. Finally, fact sorting is performed to obtain pathogen specific T cells. The evening before the oocyte enucleation procedure.
Prepare four, five, and seven micrometer piso injection needles using a syringe to retrograde load them with mercury. The next day, begin the somatic cell nuclear transfer protocol by collecting oocytes, then using a mouth pipette, transfer them into drops of K-S-O-M-A-A medium in a Petri dish. In the lid of the Petri dish.
Prepare aate for performing somatic cell nuclear transfer or SCNT by placing about 10 drops of PVP. About 10 drops of HCZB containing five micrograms per milliliter of cyto B and about 10 drops of HCCB containing 2.5 micrograms per milliliter of cyto B.This plate will be referred to as the S-C-N-T-A plate. Place the S-C-N-T-A plate on the stage of an inverted compound microscope.
Next place a seven micrometer enucleation needle into the micro manipulator and wash it by withdrawing into discarding PVP solution five to 10 times. Then transfer 10 to 30 metaphase two arrested cytes from the Petri dish into one of the drops of HCCB with five micrograms per milliliter. Cyto B on the S-C-N-T-A plate incubate for five minutes at room temperature during the incubation, line up the oocytes horizontally.
After five minutes have passed. Use the enucleation needle to place an oocyte in front of the holding needle. Using a micro injector, apply a vacuum to fix the oocyte to the micro pipette holding needle.
Next, using the enucleation needle, turn the oocyte until the chromosome spindle complex, which appears hyper transparent in comparison to the op plasma is in the three o'clock position, initiate a pizo pulse by pressing the pedal to penetrate the zno lucita. A pizo pulse is a mechanical pulse that travels longitudinally and vibrates the tip of the nucleation needle. The tip vibrations aid in drilling through the zop lucita.
Once through the zona palita, place the opening of the nucleation needle adjacent to the metaphase two spindle and apply the vacuum. The chromosome spindle complex will slowly move into the needle. Once the needle is removed, the membrane should seal itself.
Finally, using a mouth pipette transfer the enucleated cytes back into the K-S-O-M-A-A drops. Then using the same pipette, wash them by transferring them from drop to drop of KSOM AA To perform nuclear transfer exchange the seven micrometer enucleation needle on the micro manipulator with a four to five micrometer nuclear transfer needle. Wash the needle with PVP as before.
Place the CD eight positive T cells isolated earlier into a drop of PVP on the S-C-N-D-A plate. Mix well and incubate for at least 10 minutes at room temperature. Next, transfer 10 to 30 ENUCLEATED cytes into a drop of HCZB with 2.5 micrograms per milliliter.
Cyto B on the S ct. A plate incubate for five minutes at room temperature. During the incubation, use the nuclear transfer needle to line up the cytes horizontally using the injection needle.
Pick up a donor CD eight positive cell from the PVP medium and aspirate it in and out until the outer membrane has broken down. If necessary, apply a pizo pulse fragments of the membrane and cytosol should be visible. Draw up the nucleus into the needle.
Then continue to pick up nuclei until there are 10 to 30. Next, move to the drop containing the aligned enucleated cytes. Apply the vacuum to fix one oocyte to the holding needle.
Then place the NU nuclear transfer needle adjacent to the zop lucita with the nuclei oriented away from the opening. Apply pizo pulse until the needle has gone through the zop lucita. Push the micro manipulator to carefully move one nucleus to the tip of the needle.
Push the needle into the oocyte until the tip of the needle is two thirds of the way inside. Apply a small negative pressure so that a little bit of the oocyte membrane is in the needle. The next step is very critical.
Since it is the only time when the oocyte is actually open. Apply a single ZO pulse. Then apply positive pressure to push the nucleus out of the needle.
Carefully pull back the needle so that the tip is about one third of the way in the oocyte, and then apply negative pressure and continue to pull back the needle. To seal the oocyte. Repeat this step with each oocyte.
After all of the oocytes have received a nucleus, transfer them to the Petri dish and wash them by transferring them from drop to drop of KS OM AA media incubate at 37 degrees Celsius for at least 30 minutes. Following the incubation, transfer the SCN T embryos into drops of calcium free activation media containing strontium chloride. Strontium chloride activates the oocyte by inducing the calcium oscillations that would occur similarly during fertilization.
Incubate for six hours. If wanted, ADD TSA, which is an unspecific inhibitor of histone diacetyl assis to the activation media In order to improve efficiency following activation, examine the embryos under the inverted microscope to determine the amount of pseudo pronuclei or PPN positive SCNT embryos. The PPN can be identified based on their appearance.
They look like eggs. Sunny side up wash and culture. The embryos and K-S-O-M-A-A drops for three and a half days at 37 degrees Celsius until they have reached the blast assist stage.
In the beginning, you should focus on correctly performing the procedures we have just shown you for nucleation and NU nuclear transfer. Once you've mastered these techniques, the following procedures are easier to manage The embryonic stem or ES cells used. Here were derived using standard techniques.
These should be prepared on the day of blast assisted injection, three and a half days post cotus. Prepare the lid of a sterile bacterial dish by plating drops containing PVP and HCZP. Then prepare a 15 micrometer injection needle for the piso pulse as before.
Next, place the injection needle in the micro manipulator and wash it with PVP transfer. 10 to 30 blast assists into a drop of HCZB. Then place five to 50 microliters of the ESL suspension into another drop of HCZB.
There should be enough ES cells so that they can easily be picked up, but should not be crowded. Next, pick up 30 to 100 ES cells with the injection needle. Move to the drop with the blast assist.
Align the blast assist horizontally and fix one with the holding needle by applying vacuum. Use the injection needle to turn the blast assist until the inner cell mass is at nine o'clock. Place the injection needle at three o'clock.
Make sure that there are no ES cells at the tip of the needle, and apply a piso pulse until the injection needle penetrates through the zop lucita and the trifecta into the blastic seal. Release five to 15 ES cells so that they stick to the inner cell mass. Continue until all blast assist have been injected after injections are finished.
Washed twice in K-S-O-M-A-A and proceed to surgical embryo Transfer into pseudo pregnant females place and anesthetized pseudo pregnant female recipient mouse in the surgical area. Using an electric shaver, remove the hair from the lower right quadrant. Disinfect the area using an iodine based solution and 70%ethanol under a dissecting microscope.
Use scissors to open the skin, then place closed scissors between the skin and peritoneum and open them to separate the peritoneum from the skin. Locate the ovary through the peritoneum, which should appear as a red spot. Then use scissors to open the peritoneum in that region.
Use forceps to carefully grab the UCT or the surrounding fat and pull out the uterus using a capillary connected to a mouth pipette. Pick up a group of about 10 blast assists holding the oviduct and uterus with forceps. Use a needle to punch a small hole in the uterus.
Then insert the capillary with the blast assist into the lumen of the uterus and release them. Push the uterus back into the abdominal cavity. Locate the edges of the peritoneum and close it with a few stitches.
Close the skin of the mouse with a few staples. Administer pain medication subcutaneously. Then transfer the mouse back into its cage under a heating lamp.
Monitor the mouse's breathing until it wakes up. Following enucleation and somatic cell nuclear transfer. About 70%of the embryos were positive for pseudo pronuclei.
As shown in this figure. Treatment with TSA for either six or 10 hours had no effect. After three and a half days in culture, five to 10%of embryos developed into blast assist.
Again, the addition of TSA had no effect on the rate of blast assist development. The SCNT blast assist were then used to derive embryonic stem cells. TSA significantly improved the rate at which ESL were derived from blast assist.
The S-C-N-T-E-S cells are used to generate chimeric mice. Blood is drawn from these mice and analyzed for the presence of specific T cells using facts analysis as shown here. When blood is stained with anti CDA antibody and specific tetramer, a double positive population can be identified.
Indicating that the donor cell used for SCNT was of correct specificity. I have just shown you how to perform somatic cell LU transfer, blast assist injections and embryo transfer. The most important point to consider is the purity of your T-cell or B cells.
Once you have learned how to perform somatic transfer and your embryos either blast stage, you can choose between transferring them directly into pseudo pregnant females or to use them to derive embryonic stem cells and to generate chimeric mice.
