The overall goal of this procedure is to obtain developing embryos derived from OO sites that were manipulated in culture. This is accomplished by first isolating donor oo sites from one female and manipulating them as desired in culture. The second step of the procedure is to stimulate these cytes to mature using progesterone and to inject prospective host females with gonadotropin to stimulate egg laying.
The third step of the procedure is to stain the donor cytes with vital ties and prepare a female to serve as the host. The final step of the procedure is to surgically transfer the donor cytes to the body cavity of the host, and several hours later recover and fertilize the transferred eggs. Ultimately normally dividing embryos derived from the experimentally manipulated donor.
Cytes can be obtained and analyzed just like normal embryos. Hi, I am Doug Houston from the University of Iowa. Today we're at the Cold Spring Harbor Xap course to show you how to perform the host transfer procedure.
We use this procedure in our laboratory to study the roles of maternal gene products and determine the roles in early zap development. So let's get started. Before beginning surgery, be sure that the surgical area is disinfected and that fresh media and sterile tools are ready.
Grasp a suturing needle in the needle holder in the desired orientation. Place an anesthetized female frog on its back on a damp surgical wipe. Using small iris scissors, make a one centimeter incision in the skin in the lower part of the abdomen, lateral to the midline.
Do not cut into the abdominal muscle. Use forceps to lift the muscle layer and surrounding white colored fascia. Use the iris scissors to make a single swift cut straight down through the muscle.
Extend the incision to one centimeter exposing the ovary. Use forceps to grasp the ovary and pull out a lobe. Use scissors to trim the lobe off at the level of the body wall.
Place the tissue in a 90 millimeter dish containing oocyte culture medium or OCM immediately assess ovary quality under a dissecting microscope. A few cytes can be defoliated with forceps. If the cytes are firm and of uniform size and coloration, remove the desired amount of ovary tissue from the frog and place in a 90 millimeter Petri dish of OCM.
Close the incision. Insert the needle a few millimeters lateral to the incision, making sure to pass through both muscle and fascia. Use forceps to manipulate the needle, taking care not to pull the tail of the suture all the way through.
Close the wound with a simple interrupted pattern of suturing and surgeons square knots. The technique is described in detail In the accompanying text. Repeat the suturing with the skin layer.
Rinse off the female with deionized water and place her in a recovery bucket filled with cool water. Immediately after finishing surgery, use scissors to subdivide the ovary. Cut open individual lobes, flatten them and cut into two centimeter square pieces.
Rinse with clean, medium and use forceps. To transfer five to six pieces to a fresh 90 millimeter dish of OCM to isolate individual cytes. Begin by transferring one of the pieces to a small dish of medium and bring to a dissecting microscope.
Use watchmaker's forceps to manually ate three to 500 oocytes. Grasp the connective tissue layer surrounding a fully grown stage six cyte near the stock with a second pair of forceps. Grasp the ovary adjacent to the oocyte with the first pair of bps.
Lightly pull across the oocyte to tear open the follicle layer. Gently tease out the cyte from the tissue successfully. Defoliated cytes have no visible blood vessels and are floppier than cytes with intact follicles.
Use a cotton plugged fire polished pipette to transfer groups of 100 to 150 cytes to 60 millimeter culture dishes containing eight milliliters of OCM. Store the oocytes in their dishes at 18 degrees Celsius. The oocytes can now be micro injected as desired with antisense oligonucleotides or mRNAs according to any number of protocols, then cultured in OCM in an 18 degrees Celsius incubator.
Use oocytes for transfer within 96 hours of isolation on the evening prior to performing the transfer. Incubate the necessary cytes with progesterone to mature them at 16 microliters of one millimolar progesterone to the medium containing the cytes for a final concentration of two micromolar. Incubate for 10 to 12 hours at 18 degrees Celsius on the same evening, inject three to five females with 1000 units of human chorionic gonadotropin per frog to induce egg laying.
Leave females at room temperature or 18 degrees Celsius overnight on the morning of the transfer. Check under the dissecting scope to ensure that the cultured cytes have undergone maturation. Haw the vital dyes and spin at greater than 10, 000 Gs for five minutes.
To reduce background from debris, add 80 microliters of the appropriate vital dye to each experimental group of oocytes. Incubate room temperature for 15 minutes on a rocker. Transfer the colored eggs to a 90 millimeter dish of fresh o cm and swirl briefly to wash them.
Make a pipette to use for transferring the cytes. Use a diamond pencil to score a past your pipette about two centimeters below the tapered portion and break it cleanly. This should create a pipette with a 1.5 millimeter bore just large enough to accommodate an oocyte fire.
Polish the tip until the edges are smooth without closing the tip. Oocyte transplantation. Surgery should be performed as rapidly as possible.
Make an incision as before in an anesthetized female laying good quality eggs. This incision should be the minimum length required to just fit the bore of the transfer. Pipette less than one centimeter.
Swirl the dish of experimental oocytes to collect them in the center of the dish. Use the prepared pasture pipette to suck up as many as possible. Allow the oocytes to settle into the end of the pipette to minimize the amount of liquid transferred to the frog.
Use forceps to grasp and elevate one flap of muscle and fascia. Insert the tip of the pipette into the incision and slowly allow the cytes to trickle into the body cavity. Repeat the process until all oocytes have been transferred.
Do not release the muscle layer to prevent the oocytes from spilling out of the incision. Use forceps to grasp both sides of the incision and bring them together, allowing the cytes to settle into the body cavity. Begin suturing the muscle and fascia layer as before taking care to keep upward tension until the first knot is tied.
Suture the skin. Rinse the frog with deionized water and allow her to recover as before. The frog should begin laying eggs normally after the operation beginning two to three hours after surgery.
Recover eggs by normal squeezing every half hour until the female stops laying to a dish of collected eggs. Add four milliliters of a suspension of freshly collected sperm in one XMMR and incubate at room temperature for four minutes. Flood and rinse the eggs extensively with room temperature.
Point one XMMR incubated room temperature to allow activation and cleavage. Use cysteine at any point to remove the jelly codes. The transferred embryos can then be sorted into separate dishes depending on color and cultured as normal embryos.
Typically, more than two thirds of the transferred eggs will activate within minutes of fertilization showing robust cortical contraction at the animal pole. In most experiments, over half of the transferred eggs will undergo normal first cleavage. Sometimes cleavage will be delayed 20 minutes or so relative to the host eggs in our hands.
30 to 60%of the transferred embryos go on to gastro light normally and will survive past the neural stage. The transferred embryos will retain their vital dye color through many days of development. Embryos derived from unin injected control cytes will appear normal as expected.
By contrast. Embryos derived from oocytes injected with beta catine and antisense oligos, for example, will have a centralized appearance. We've just shown you how to obtain embryos from cytes manipulated in culture.
When performing this procedure, it's important to start with healthy full grown donor oocytes and to use host females with high quality eggs. Also, the success of the method tends to correlate with the speed with which you isolate oocytes and perform the surgeries. So it may take some practice before getting consistent results.
So that's it. Thanks for watching and good luck with your experiments.
