Intraductal Injection: Delivering Injection Mix into the Ducts of the Mouse Mammary Gland
Transcrição
An intraductal injection is a technique of delivering an injection mix that may contain cancer cells, expression vectors, or a therapeutic agent supplemented with a tracer dye into ducts of the mammary gland.
For this procedure, load a syringe with the injection mix. Next, place an anesthetized mouse ventral side up on a surgical bed. Make an incision on the skin between the inguinal or fifth pair of mammary glands. Carefully separate the skin flap from the parietal peritoneum to visualize the ductal tree.
Using forceps, lift the nipple and carefully cut its exterior part to view the duct opening. Use a tweezer to lift the skin flap's edge, and gently insert the syringe needle into the nipple to deliver the injection mix. Monitor the movement of the tracer dye into the mammary ductal tree. The dye spreads out in the ductal tree without leaking into the stroma, indicating a successful injection.
Wait for a few seconds to avoid spillage, then slowly remove the needle from the injection site. Close the incision with the wound clip. In the following protocol, we will perform intraductal injection to deliver Cre expressing adenovirus into the mouse mammary gland.
Use aseptic techniques throughout the surgical procedure. Make an incision site on the skin at a length of approximately one centimeter between the two fourth inguinal mammary glands. Carefully separate the skin flap from the parietal peritoneum, so as to visualize the mammary ductal tree.
Carefully hold the nipple with watchmakers forceps to remove the exterior nipple without cutting any nearby skin using a micro-dissection scissor. Load approximately three to five microliters of Cre adenovirus injection mixture into a 25 microliter Hamilton syringe with a 33 gauge metal hub needle affixed.
Estimate the volume of the injection mixture in the syringe based on the blue dye included in the mixture. Gently hold the edge of the skin flap with a fine curved tweezer and inject the Cre adenovirus injection mixture slowly into the nipple while monitoring the spreading of blue dye into the mammary ductal tree. Maintain the injection rate as slow as possible to avoid damage to the ductal lumen.
A successful intraductal injection is indicated by injected fluids spreading throughout the entire ductal tree without leaking into the stromal compartment. Gently withdraw the needle from the nipple to avoid any leakage of the injected fluid. Examine the distal side of the mammary gland or the surrounding area of the injected nipple.
Dye diffusing into the nearby stroma, as shown here, indicates a mammary fat pad injection rather than a successful intraductal injection. Close the surgical wounds and the skin with wound clips.
Remove the mouse from the anesthesia and place it on a heating pad inside a clean cage for recovery. Monitor the development of the mammary tumor as described in the text protocol.
