Our protocol permits an efficient identification of gene or gene combinations that block cancer invasion in viewer. over culture does not require advanced animal facilities for maintenance. As a whole, as a generic or general special libraries can be screened in a matter of several weeks in viewer.
This over screening system will help researchers discover novel therapeutic gene targets or gene targets combinations that it can be used to block cancer metastasis. For IV injection, concentrate cells to 0.5 to 1 million cells per milliliter, using ice cold 1X PBS to dilute or re-suspend the cells. Expect approximately one milliliter of cell suspension will be needed for every 10 embryos.
To assemble the injection apparatus, mount a needle onto the syringe, and then extend the syringe needle with a three to five centimeter long piece of tubing. Break the tip of the borosilicate needle using fine forceps. Load the syringe with 50 to 200 microliters of the cancer cell suspension.
And insert the borosilicate needle into the tubing. Inspect the needle for cell clogging and air bubbles. If cell clogging is observed within the capillary, remove the capillary, re-suspend the cells and replace it with a new capillary.
Use the plunger to push out any bubbles. Remove the cover lid and transfer the embryo under the stereoscope. Identify the appropriate vein to be injected on the CAM surface;which is normally only slightly wider than the diameter of the borosilicate needle tip and located midway between the embryo and the weighing dish wall.
It is generally easier to inject at the point immediately adjacent to the vein bifurcation. Press the tip of the needle against the blood vessel wall and apply gentle pressure in the same direction as the blood flow. If necessary, use a cotton swab to help anchor or stabilize the vessel that is being injected.
Gently depress the syringe plunger for 2 to 10 seconds until the desired volume is injected. Discard the embryo if excessive bleeding or clear liquid accumulation appears at the injection site. Remove the needle from the CAM and gently dab the injection site with a cotton swab to remove any blood or excess cancer cells.
Cover the injected embryo in the weighing dish with the lid and return it to the incubator. Repeat the procedure until all embryos are injected. Visually inspect the embryos for bacteria or mold contamination or death.
And discard contaminated embryos according to laboratory disposal procedures. Ensure that metastatic colonies appear uniform in shape during the first one to three days post-injection. And identify invasive or noninvasive metastatic colonies at days four to five post-injection.
At day five post-injection, remove the embryos from the incubator and inspect and locate the embryo CAMs for metastatic colony distribution. Under the dissection microscope, gently pull the CAM tissue that contains the metastatic colony of interest upwards using fine forceps, and cut it off with surgical scissors. Transfer the CAM tissue into an empty, sterile 1.5 milliliter tube and close the tube lid.
Repeat the excision procedure until all the colonies of interest are collected into separate tubes. Gently mince the CAM tissue in a micro centrifuge tube, using a separate sterile 18 gauge needle for each colony. Add 100 microliters of 1X collagen-A solution and incubate for 30 minutes at 37 degrees Celsius.
Spin down the cells and CAM tissue at 300 times G for five minutes at ambient temperature. Aspirate the collagen-A solution and re-suspend the cells incomplete media for the cell line of interest. Then spin the cells and tissue again.
Re-suspend cells and tissue pieces in one milliliter of complete media and selection factor, if any. Then transfer into a single 12-well tissue culture dish well. For the next one to three weeks, monitor the cancer cells daily for growth and contamination.
When the cells reach 70 to 80%of confluency transfer them into a larger volume culture dish. Proceed to sequencing or the next round of selection, as soon as adequate cancer cell numbers are reached. Embryos displaying a buildup of cancer cells due to unsuccessful injection, can be seen in most of the capillaries.
When the optimal cell concentration and duration of injection is achieved, day five post-injection transduced cells should produce a wide variety of colony phenotypes, with the majority of the colonies invasive as determined by the cancer cells appearing scattered in the CAM tissue. Attention should be paid to the metastatic colonies that appear compact and are located far enough from neighboring colonies that they can be excised with forceps and scissors and one piece of CAM tissue. An isolated positive screen hit should display the compact colony phenotype upon re-injection.
For measurement of the cancer cell blood vessel contacts, attention should be paid to the vascular wall stain brightness and the appropriate amount of lectins should be injected for the vascular wall signal. When attempting this protocol avoid under or over injection and remove excessive bleeding or liquid buildup. Several novel genes that are driving cancer cell invasion in viewer have been identified using this technique.
