Part 3: Host Transfer Technique
Protocol submitted by Janet Heasman [1]
Once a suitable ODN has been identified, the effect of depletion of the mRNA should be tested in developing embryos. To observe the effects of the ODN, oocytes depleted of the target mRNA are fertilized by the use of the host transfer technique (see fig.2 for a schematic representation)
Choose an ovary. The major determinant in the success of this technique is the quality of oocytes injected and transferred. Unfortunately, there is no sure test of oocyte quality. Therefore we routinely anaesthetize several donor frogs consecutively (Xenopus 1, Xenopus Express, Nasco) and remove small pieces of ovary in a sterile fashion, via an abdominal incision (for further detail see 'transferring the oocytes into an egg-laying female frog' below). Frogs are selected to donate ovary based on two criteria. First, it has been our experience that frogs that do not look healthy and plump do not yield 'good' ovary, so that we pick healthy, plump, mature females to operate on. Second, frogs which have been injected with human chorionic gonadotropin (HCG) in the previous 10 weeks are not used, either.
The ovary itself is not used if there are very few full grown oocytes, oocytes with small cavitations in the animal hemisphere, or many oocytes that are flaccid or wrinkled (Heasman et al., 1991). Oocytes with conspicuous equatorial bands often fertilize well (but not always), although these bands may not be obvious on palely-pigmented oocytes (Heasman et al., 1991). One test that may be used to determine the suitability of an ovary is to defolliculate a small number of oocytes and stimulate them to mature using a final concentration of 1 µM progesterone in OCM. This test is useful since maturation is a necessary step for fertilization and oocytes which do not mature well (less than 80%) will not fertilize well. Care should be taken to defolliculate only the largest subset of healthy oocytes in order to ensure the best chance of fertilization and uniformity of embryonic response to ODN.
Remove and subdivide the ovary into separate dishes Having chosen a suitable ovary, enough is withdrawn from the anaesthetized frog via the abdominal incision for the experiment. The number of 'good' oocytes per lobe of ovary varies greatly, but 2-3 lobes will often yield about 500 oocytes. Ovary is washed in OCM to remove blood, and is then cut into small pieces (generally about 1cm x 2cm) and cultured in OCM at 18 degrees. Care is taken not to overcrowd the dishes with ovary, as the tissue is highly metabolically active (6-7 pieces per 90 mm dish). In these conditions ovary can be maintained in culture 2-4 days. Before subdividing ovary, however, the anesthetized frog should be sutured (for further detail see 'transferring the oocytes into an egg-laying female frog' below) and put in a clean bucket containing tap water and 3 ml/10 gallons of Amquel (Kordon).
Defolliculate and inject. To study the function of the test mRNA large numbers of defolliculated oocytes are required (approximately 500 for a large experiment). We manually defolliculate oocytes using jewelerÕs forceps (#4, American Surgical Instruments Corporation), as collagenase-treated oocytes do not fertilize. In an attempt to keep the media as sterile as possible while still making manual defolliculation technically feasible, we wipe down the watchmakerÕs forceps with 70% ethanol before use. Also, in a variation from previously published technique (Heasman et al., 1991), we incubate the defolliculated oocytes in dishes of OCM which have not been previously agarose-coated. After defolliculation, modified ODNs are diluted with sterile filtered distilled water to a concentration less than 0.5 ng/ nl. Often ODNs are diluted to 0.2 and 0.4 mg/ml, and are spun at 4 degrees and 20,000 rpm for 10 mins. Oocytes are injected in the equatorial zones of oocytes in OCM, as superficially within the oocyte as possible in batches of 70-100. Initially, a typical experiment includes batches of 70-100 uninjected or 4 ng of sense ODN only as well as 1, 2, 3 and 4 ng of modified ODN. We limit ourselves to 5 batches of oocytes per experiment due to the limitation of vital dyes (Appendix B). After initial successful underexpression experiments have been completed, a dose is found which causes an effective depletion. Then that dose and one other are usually injected so that a phenotype may be observed even if there is variability in the response of the oocyte to ODN. Oocytes are all incubated at least 24 hours in order to allow the ODNs to decay.
Progesterone oocytes and inject frogs with human chorionic gonadotropin (HCG) Oocytes are prepared for fertilization by adding a final concentration of 1 µM progesterone (from to the OCM, which matures them, and then culturing them at 18 degrees for 12 hours. Host frogs are stimulated the night before with human chorionic gonadotrophin (1000 U, Sigma) so that they will begin to lay eggs 8-12 hours later at room temperature. Typically progesterone treatment and frog HCG injection are carried out at 9-10pm for a 9 am transfer the following day.
Freeze some oocytes for RNA analysis and stain the other oocytes with vital dyes. Before transferring the oocytes to a host frog, a small number of oocytes are frozen for Northern analysis and the rest are colored with vital dyes (see Appendix B). Dyes are added directly to the OCM containing the oocytes. We use 100 µl of red, blue or brown dyes in 8 ml of OCM. Green is obtained by adding 100 µl of blue and brown, and purple (mauve) by mixing 100 µl of blue and red. Oocytes are colored for approximately 15 mins on a rocking platform, and then washed in a large dish of OCM.
Transfer the oocytes into an egg-laying female. A host frog that has just begun to lay eggs is selected for oocyte transfer. The newly laid eggs are examined to check that the host is not laying eggs that are in heavily jellied strings, or that are clearly dead. The host is then anaesthetized using MS222 (3-aminobenzoic acid ethyl ester methanesulfonate salt, Sigma). As soon as the frog is no longer responsive to touch on the bottom of the jaw, a small incision is made through the skin in the lower half of the anterior abdominal wall, to one side of the midline. The connective tissue and muscle layer under the skin are also opened with one cut using small scissors, thus making an incision into the coelomic cavity. The incision is large enough to allow the entrance of the end of a pasteur pipette, and about half the size of the incision which is used to remove ovary. The sterile pasteur pipette is marked with a diamond pencil so that when the end of the pipette is broken off, the diameter of the end of the pipette is slightly larger than a full-grown oocyte. The pasteur pipette is cut to a diameter slightly larger than an oocyte with a diamond pencil and flamed to produce smooth surfaces. Then the pipette is used to transfer all of the experimental oocytes into the coelomic cavity.
It is important in any operation on the frog to hold one side of the incision in the forceps while the oocytes are introduced with the other hand. If the forceps hold the edge of the incision a small distance above the frog, valuable time will not be wasted looking for the incision and valuable oocytes will not be lost as they are expelled out of the coelomic cavity. Care is also taken not to introduce air, or large excesses of OCM.
The oocytes can be injected into any part of the coelomic cavity, as they will be moved into the oviducts by the ciliary action of the coelomic epithelium. We have introduced as many as 700 oocytes into one host frog. The layers of the body wall are then sutured (45 cm 4-0 black braided silk, C3, cutting 3/8 circle, 13 mm, Ethicon) using 2 or 3 stitches to sew together the muscle and overlying fascia, and 2 or 3 stitches are used for sewing the skin, taking care not to sew skin and muscle or skin and fascia together. The sutures are applied using a hemostat. The frog is returned to clean water and recovers from the anesthetic within 10 -20 minutes. Care should be taken not to overexpose frogs to anesthetic, i.e. to carry out the transfer of oocytes as soon as movement has ceased, and to use sterile technique as much as possible (e.g. dip instruments in ethanol and flame) when operating on the frog.