用いた機能クローニング

The overall goal of this procedure is to demonstrate a Xenopus oocyte, an animal cap system, suitable for the identification of gene products capable of inducing a response in competent ectoderm. This method can help answer key questions in the field of developmental biology such as what genes are necessary to illicit an inductive response. The main advantage of this technique is its use of the Xenopus oocyte, as an expression system, to produce and identify inducers acting upon a target tissue, or even genes acting upstream of inducers.

Visual demonstration of this method is extremely useful, as the dissection and recombination steps are highly stage dependent and require a refined technique. After collecting ovarian tissue from anesthetized female frogs, tear the tissue into small pieces, each containing approximately 10 to 20 oocytes. And transfer these fragments, first to fresh OR2-solution, and then to OR2-containing 2.0 milligrams per milliliter collagenase A.Continue by gently agitating the tissue-containing mixture on a shaker for one hour.

Then transfer the fragments to fresh collagenase solution, and agitate for an additional hour. Expect to observe deflocculated oocytes at the end of this treatment. Proceed to wash the oocytes 10 times in complete OR2, and then twice in oocyte culture medium, abbreviated as OCM.

Afterwards, place the oocytes in fresh OCM, and visually inspect them under a dissection microscope. Isolate those at stage six by discarding the smaller, immature oocytes. Next, place the oocytes in an agarose-coated Petri dish containing OCM, and maintain them at 18 to 20 degrees Celsius until injection.

To prepare for microinjection, place glass capillary tubing into a needle puller, pull it, and then break the tips off the glass to generate needles approximately 20 micrometers in diameter. On a compound microscope, measure the needle tips with a calibrated ocular micrometer. Next, push clay into a uniform layer on the bottom of a 35 by 10 millimeter Petri dish.

And then create parallel grooves in this layer, using a mall probe-like tool. Fill the clay-lined dish with approximately 2 milliliters of 3%Ficoll in 1x modified Barth's saline, or MBS. And then transfer the oocytes to it, using a wide-bore pipette.

Position the oocytes in the groves so they will be held in place during the subsequent microinjection. Using a microinjector, fill a needle with approximately two microliters of a previously generated pool of mRNA and adjust the balance to produce slight positive pressure, which will prevent oocyte cytoplasm from being drawn into the needle during injection. Then, inject each oocyte with 20 nanoliters of RNA in the equatorial region.

Afterwards, leave the oocytes in the Ficoll MBS for one hour, and then gently transfer them to 1x MBS. Proceed to incubate the oocytes for eight to 24 hours at 20 degrees Celsius. To begin the animal cap assay, gather 3/4x normal amphibian medium, abbreviated as NAM solution, fine forceps, a hairloop, previously injected oocytes, and xenopus embryos at stage 11 to 11.5.

Then, break apart glass cover slips into fragments approximately one millimeters by two millimeters in size, and pass these pieces through a flame until their edges polish and drew. Next, press clay into a single layer in a dish, as previously described. And use a mall probe to create individual cup-shaped impressions in this coating.

Proceed to fill the dish with approximately two milliliters of 3/4x NAM. And to it, transfer the injected oocytes, positioning several so that each is immobilized in a single indentation. To ready the embryos, transfer them in a dish containing 3/4x NAM solution.

Select a subset of gastrula to be used as staging controls for the age of the ectoderm, and transfer them to another dish containing the same solution. And set this plate aside. For embryos that will be used in the animal cap assey, remove their vitelline membranes with two pairs of fine forceps.

Afterwards, transfer the gastrula to the clay-lined dish with the oocytes. To begin, cut the animal caps from the embryos using two pairs of fine foreceps, being careful to avoid the equatorial tissue. To generate recombinants using the first method, position an animal caps so that the inner surface contacts the animal hemisphere of an oocyte, already positioned in an indentation.

And repeat this process for several of the injected oocytes. To ensure that these recombinants are held together, place a curved glass coverslip fragment on top of each of them, and apply downward pressure until the animal is flattened and the coverslip contacts the clay. To generate recombinants using the second method, place animal caps and oocytes together in empty individual indentations in the clay.

And ensure the animal caps inner surfaces are facing the oocyte. Then, secure the two tissues together using small extensions of clay. Once multiple recombinants have been generated using either method, culture then and the set-aside control embryos at 20 degrees celsius.

When the control embryos reach the desired stage, remove the coverslips from the recombinants and isolate the animal cap ectoderm using forceps and a hair loop. Proceed to fix both ectodermal fragments and control embryos in MEMFA for one hour. And transfer them to ethanol and store this tissue at negative 20 degrees celsius.

Here, sets of oocytes were injected with pools of mRNA corresponding to progressively smaller numbers of clones or colonies, and then, cultured with animal caps. Subsequently, the number of animal caps in each set expressing foxe3, a marker normally observed in presumptive lens ectoderm from early neural plate stages to vesicle formation, and use here, to evaluate lens inducing capability, was determined. This method identified in nuclear factor encoding gene, ldb1, which can produce a lens-inducing response in 50 out of 179, or 28%of animal caps assessed.

Shown here, our in situ hybridization images, depicting foxe3 signal, indicated by arrow heads, in animal caps cultured with ldb1 injected oocytes from roughly stage 11 to stage 23. No foxe3 expression is observed in corresponding control animal caps placed on uninjected oocytes. When sections were generated from these animal caps, foxe3 expression was observed in both the inner and outer ectodermal layers.

Expression in the inner layer is characteristic of a lens response. Or, as signaled in both layers, is indicative of other structures such as the olfactory placode. Following this procedure, methods like immunohistochemistry, in situ hybridization, or direct detection of a fluorescent reporter gene, can be performed in order to assess an inductive response in the animal cap ectoderm.